Non-psychotropic cannabinoids

ABSTRACT

Novel non-psychotropic cannabinoids are disclosed and pharmaceutical compositions comprising these novel compounds are described for preventing neurotoxicity, neuroinflammation, immune or inflammatory disorders comprising as active ingredient the stereospecific (+) enantiomer, having (3S,4S) configuration of Δ 6  tetrahydrocannabinol type compounds. The compositions are particularly effective in alleviating and even preventing neurotoxicity due to acute injuries to the central nervous system, including mechanical trauma, compromised or reduced blood supply as may occur in cardiac arrest or stroke, or poisonings. They are also effective in the treatment of certain inflammatory disorders and chronic degenerative diseases characterized by neuronal loss and chronic pain including neuropathic pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of the US national stage designationof International Application PCT/IL01/00571 filed Jun. 22, 2001, whichclaims priority to US provisional application No. 60/222,467 filed Jul.28, 2000, and Israeli patent application 136946 filed Jun. 22, 2000.

FIELD OF THE INVENTION

The present invention relates to a family of novel non-psychotropiccannabinoids, and to pharmaceutical compositions containing them, whichare useful for preventing or alleviating neurotoxicity and inflammation.Said pharmaceutical compositions comprise as their active ingredient thestereospecific (+) enantiomers, having (3S,4S) configuration, ofΔ⁶-tetrahydrocannabinol (THC) type compounds of general formula (I), asdefined hereinbelow.

BACKGROUND OF THE INVENTION

The identification of tetrahydrocannabinol (THC) as the active principleof marijuana (Cannabis sativa) prompted medicinal chemists to developnumerous cannabinoid analogs (reviewed by Barth, in Exp. Opin. Ther.Patents 8:301-313, 1998). These novel compounds were designed to exhibitthe beneficial properties of THC without the accompanying psychotropiceffects, which limit its therapeutic utility. Potential therapeuticapplications have classically included known attributes of marijuanaitself such as anti-emesis, analgesia, antiglaucoma and appetitestimulation. More recently recognized roles for non-psychotropiccannabinoids are as neuroprotective and anti-inflammatory agents.

Neuroprotective Activity

Chronic degenerative changes, as well as delayed or secondary neuronaldamage following direct injury to the central nervous system (CNS), mayresult from pathologic changes in the brain's endogenous neurochemicalsystems. Although the precise mechanisms mediating secondary damage arepoorly understood, post-traumatic neurochemical changes may includeoveractivation of neurotransmitter release or re-uptake, changes inpresynaptic or postsynaptic receptor binding, or the pathologic releaseor synthesis of endogenous factors. The identification andcharacterization of these factors and of the timing of the neurochemicalcascade after CNS injury provides a window of opportunity for treatmentwith pharmacologic agents that modify synthesis, release, receptorbinding, or physiologic activity with subsequent attenuation of neuronaldamage and improvement in outcome. A number of studies have suggestedthat modification of post-injury events through pharmacologicintervention can promote functional recovery in both a variety of animalmodels and clinical CNS injury. Pharmacologic manipulation of endogenoussystems by such diverse pharmacologic agents as anticholinergics,excitatory amino acid antagonists, including specifically NMDA receptorantagonists, endogenous opioid antagonists, catecholamines, serotoninantagonists, modulators of arachidonic acid, antioxidants and freeradical scavengers, steroid and lipid peroxidation inhibitors, plateletactivating factor antagonists, anion exchange inhibitors, magnesium,gangliosides, and calcium channel antagonists have all been suggested topotentially improve functional outcome after brain injury (McIntosh, J.Neurotrauma 10:215-243, 1993).

The pathogenesis of a diverse group of neurological disorders has beenlinked to excessive activation of excitatory amino acid receptors. Thesedisorders include epilepsy, focal and global ischemia, CNS trauma, andvarious forms of neurodegeneration including Huntington's chorea,Parkinson's disease and Alzheimer's disease. There has been extensiveeffort invested in the development of excitatory amino acid receptorantagonists as therapeutic agents (Rogawski, Trends in Pharmacol. Sci.14:325-331, 1993 and Danbolt, Progress in Neurobiology 65:1-105, 2001).

Since no proven effective therapy for neuronal injury, or degeneration,is yet known, and, for example, stroke alone is one of the leadingcauses of death in many countries, the importance of finding suchtherapeutic NMDA antagonists is self-evident. It will be important todetermine whether certain NMDA receptor antagonists are moreeffective—or have fewer side effects—than others in specific diseasestates.

Some of the compounds of general Formula (I) are disclosed in U.S. Pat.No. 4,179,517 and 4,876,276. As disclosed in said U.S. patents, theseessentially pure synthetic (+)-(3S,4S)-THC derivatives and analogues aredevoid of any undesired cannabimimetic psychotropic side effects. Theseknown compounds have been described as having analgesic, antiemetic andantiglaucoma activity.

A particular compound of interest of Formula I, namely 1,1 dimethylheptyl-(3S,4S)-7-hydroxy-Δ⁶-tetrahydrocannabinol, is disclosed in U.S.Pat. No. 4,876,276, and denoted therein as HU-211, and subsequentlyassigned the trivial chemical name dexanabinol. HU-211 was unexpectedlydiscovered to possess neuroprotective attributes, which may be ascribedto its activity as a non-competitive antagonist at the NMDA receptor, asdisclosed in U.S. Pat. Nos. 5,284,867 and 5,521,215. Certain esterderivatives of dexanabinol are also active in neuroprotection, asdisclosed in U.S. Pat. No. 6,096,740 as are the carboxylic acidderivatives of HU-211 as disclosed in U.S. Pat. Nos. 5,538,993 and5,635,530.

Anti-Inflammatory Activity

Besides NMDA receptor blocking activity, dexanabinol and its esterderivatives were further shown to possess anti-oxidative andanti-inflammatory properties, which may contribute to their efficacy inpreventing or alleviating ischemic damage to tissues.

In addition, derivatives of HU-211 were surprisingly shown to possessimmunomodulatory potential due to their ability to inhibit TumorNecrosis Factor alpha as disclosed in U.S. Pat. No. 5,932,610.

Certain natural non-psychotropic cannabinoids, including the derivativecannabidiol, have been found to have antioxidant properties unrelated toNMDA receptor antagonism as disclosed in WO 99/53917.

Endogenous ligands of the cannabinoid receptors (Mechoulam, et al.,Endocannabinoids, Eur J. Pharmacol. 359:1-18, 1998) have been identifiedas being arachidonyl derivatives including 2-arachidonyl glycerol, andarachidonyl-ethanolamide (anandamide). Thus, these endocannabinoids arechemically related to certain metabolites in the arachidonic acidpathway.

A family of compounds known to exhibit inflammatory properties is theprostaglandins (PG). Prostaglandins are arachidonic acid metabolites,produced by the action of cyclooxygenase (COX) also known as PGHsynthase. The first step in the production of prostaglandins fromarachidonic acid (AA) is the bis-oxygenation of arachidonic acid toprostaglandin PGG₂. This is followed by reduction to PGH₂ in aperoxidase reaction. COX catalyzes both of these reactions. Two isoformsof COX have been identified, COX-1 and COX-2. Although both perform thesame catalytic activity they differ in tissue distribution, regulationand expression (Williams and DuBois Am J. Physiol. 270:G393-400, 1996).

COX-1 is constitutively expressed and appears to be involved in thephysiological production of PGs. Although COX-2 has a normal pattern ofexpression in some body tissues it is primarily an inducible form thatis expressed upon prolonged exposure to chemical mediators includingcytokines and endotoxin (reviewed in Golden and Abramson, SelectiveCyclooxygenase-2 inhibitors, Osteoartritis 25:359-378, 1999) Pain andinflammation in certain pathological processes are mediated by the COX-2dependent production of PGE₂. There is considerable interest indeveloping anti-inflammatory therapeutic strategies that block theactivity of COX-2 and the biosynthesis of PGE₂ resulting from activationof the Arachidonic acid/prostaglandin (AA/PG) biosynthetic pathway.

Attenuation of COX-2 activity is correlated with a reduction in pain,inflammation and fever. For example, the NSAIDs (non-steroidalanti-inflammatory drugs) act by blocking the COX enzymes. A reduction of40-50% in the colon cancer rate among cardiovascular patients in the USwho are given prophylactic doses of aspirin (a common NSAID) was alsoshown to be related to a decrease in COX-2 expression (Smalley andDuBois, Adv Pharmacol 39:1-20, 1997).

Therapeutic strategies that target this pathway are sought to preventand treat a variety of diseases and symptoms such as neuronaldegeneration in diseases as Alzheimer's disease or Parkinson's disease,neuronal trauma associated with seizures, brain or CNS damage,inflammation associated with rheumatoid arthritis; bone resorption andcolonic polyposis and colorectal cancer (reviewed in Lipsky, J Rheumatol26: Suppl 56:25-30, 1999). U.S. Pat. No. 5,840,746 teaches the method oftreating neurodegenerative disease by administering non-steroidal COX-2inhibitors that specifically bind to COX-2. Inflammation has also beenimplicated as part of the pathogenesis in myocardial infarction,atheroma, unstable angina and other cardiac disorders (Ross, New EnglandJ Med 340:115-126, 1999).

There is an unmet need for and it would be advantageous to have novelnon-psychotropic cannabinoid compounds that exert their effects via aplurality of mechanisms. Ideally, in addition to having said analgesic,antiemetic and anti-glaucoma activities, they would also be effectiveagainst the diseases and conditions mentioned above. The mechanismsinvoked in these pleitropic effects include their action as excitatoryamino acid receptor blockers, for example NMDA-receptor orglutamate-blockers or interaction with the glycine receptor, or asinhibitors of either the oxidative, cytokine, nitric oxide or AA/PGpathways, including the cyclooxygenase and lipoxygenase and areeffective in the alleviation and treatment of many of the abnormalstates involving said neurotransmitter or pathway mediated toxicity. Thepresent invention now provides such compounds.

SUMMARY OF THE INVENTION

The present invention relates to pharmacologically acceptablenon-psychotropic cannabinoids. These compounds act as agents that canafford neuroprotection by exhibiting anti-inflammatory activity, and/orantioxidative activity, and/or the capacity to block the AA/PG orlipoxygenase pathway, or the nitric oxide or cytokine pathways and/or toblock excitatory amino acid mediated toxicity by interaction at specificreceptors, such as glutamate receptors. In addition, the presentprovides agents that can afford neuroprotection by combinedanti-inflammatory, antioxidative and/or glutamate-receptor blockingmechanisms of action. Thus, the present invention providespharmaceutical compositions comprising as an active ingredient one ofthe non-psychotropic cannabinoids disclosed herein. These compositionsare useful for the treatment or prevention of ischemia in the CNS aswell as in other tissues such as kidney, lung, liver, heart and joints.The compositions will be neuroprotective and will be useful for theprevention or treatment of neurodegenerative disease as well as forglaucoma, pain, inflammation, and emesis.

The present invention discloses novel compounds that are effective inthe alleviation and treatment of many of the abnormal states involvinginflammation and toxicity. It will be noted that the compounds of thepresent invention may operate via diverse mechanisms to provide theneuroprotective and/or anti-inflammatory properties.

Certain embodiments of the present invention are particularly effectivein alleviating and even preventing neurotoxicity due to excitatory aminoacids, also referred to as glutamate neurotoxicity. Glutamateneurotoxicity may occur during acute injuries to the central nervoussystem (CNS), such as injuries due to prolonged seizures, compromised orreduced blood supply, deprivation of glucose supply and mechanicaltrauma. The present compositions are also effective in alleviating otherdamages to the CNS like damage resulting from poison-inducedconvulsions, including but not limited to those considered to beassociated with amino acid receptors other than that of glutamate, forexample glycine. Unexpectedly, neuroprotection is also a feature of someof the novel compounds that do not have a high affinity for the NMDAreceptor.

The compositions of the present invention may also be effective in thetreatment of certain chronic degenerative diseases that arecharacterized by gradual selective neuronal loss. In this connection,the compositions of the present invention are contemplated astherapeutically effective in the treatment of Alzheimer's disease,Parkinson's disease, Huntington's disease and amyotrophic lateralsclerosis. Surprisingly, it has been shown experimentally that morepreferred embodiments of this group of compounds can even promote nerveregeneration.

The present compositions are of special value in global hypoxic ischemicinsults, in hypoxia, alone or in combination with blood flow reduction,such as cardiac, unstable myocardial, renal and hepatic ischemias, aswell as in cases of cardiac arrest and in cases of abrupt occlusion ofcerebral arteries (stroke).

The present compositions are also particularly useful as analgesics, agenerally known attribute of this class of compounds. The presentcompositions are also of special value in inflammatory or immunediseases of 1) the nervous system, exemplified by multiple sclerosis andother autoimmune diseases, arthritis such as rheumatoid arthritis andother types of local or general inflammation, encephalitis andHIV-induced neurodegeneration; 2) the cardiovascular system, exemplifiedby myocardial infarction, coronary heart disease, restenosis of coronaryvessels and myocarditis; and 3) the pulmonary system, exemplified byasthma or chronic obstructive pulmonary disease (COPD).

The invention also provides compositions that can inhibit the AA/PGsignaling pathways that regulate or are regulated by COX-2, an examplebeing the prevention or treatment of the occurrence or growth ofgastrointestinal tumors such as colorectal cancer and colonic polyps.

The therapeutic agents of the present invention comprise novelderivatives of non-psychotropic cannabinoids.

A first embodiment of the present invention provides novel compoundsaccording to formula (I):

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer, wherein A—B indicates an optional 1(2) or 6(1)double bond,

R₁ is

A) R3 where R3 is selected from the group consisting of

a) a linear or branched, saturated or unsaturated, carbon side chaincomprising 1-8 carbon atoms interrupted by 1-3 heteroatoms; or

b) a saturated or unsaturated cyclic moiety or an aromatic orheterocyclic moiety having from 5-20 atoms comprising one or two-ringedstructures, wherein each ring comprises 3-8 carbons interrupted by 1-4heteroatoms, said heteroatoms in each independently selected from thegroup consisting of N, O, and S; wherein each ring optionally is furthersubstituted with one or more groups selected from

i) C₁₋₆ alkyl,

ii) C₁₋₆ alkoxy,

iii) C₁₋₆ alkylthio,

iv) Halo,

v) Carboxyl

vi) —CO₂—C₁₋₄ alkyl

vii) keto,

viii) nitro,

ix) a saturated or unsaturated cyclic moiety, or an aromatic or aheterocyclic moiety wherein each ring comprises 3-8 carbons interruptedby 0-4 heteroatoms, said heteroatoms in each independently selected fromthe group consisting of N, O, and S; wherein each ring optionally isfurther substituted with one or more groups selected from i)-viii) asdefined above;

B) an amine or an amide substituted with at least one substituent asdefined in R3 above;

C) a thiol, a sulfide, a sulfoxide, a sulfone, a thioester or athioamide optionally substituted with one substituent as defined in R3above; or

D) an ether —OR3 wherein R3 is as defined above;

G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR wherein R is (a′) —R″,wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal—OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or(b′) —C(O)R′″ wherein R′″ is as previously defined, and

R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain orbranched C₂-C₉ alkyl which may be substituted at the terminal carbonatom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.

For purposes of this specification C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆alkylthio are intended to include saturated and unsaturated linear,branched and cyclic structures.

Currently more preferred compounds are those wherein G is hydroxy orlower acyloxy and wherein R2 is dimethylheptyl.

The present invention further relates to pharmaceutical compositions forthe purposes set out above, comprising as an active ingredient acompound of the general formula (I):

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer, wherein A—B indicates an optional 1(2) or 6(1)double bond,

R₁ is

A) R3 where R3 is selected from the group consisting of

a) a linear or branched, saturated or unsaturated, carbon side chaincomprising 1-8 carbon atoms interrupted by 1-3 heteroatoms; or

b) a saturated or unsaturated cyclic moiety or an aromatic orheterocyclic moiety having from 5-20 atoms comprising one or two-ringedstructures, wherein each ring comprises 3-8 carbons interrupted by 1-4heteroatoms, said heteroatoms in each independently selected from thegroup consisting of N, O, and S; wherein each ring optionally is furthersubstituted with one or more groups selected from

i) C₁₋₆ alkyl,

ii) C₁₋₆ alkoxy,

iii) C₁₋₆ alkylthio,

iv) Halo,

v) Carboxyl

vi) —CO₂—C₁₋₄ alkyl

vii) keto,

viii) nitro,

ix) a saturated or unsaturated cyclic moiety, or an aromatic or aheterocyclic moiety wherein each ring comprises 3-8 carbons interruptedby 0-4 heteroatoms, said heteroatoms in each independently selected fromthe group consisting of N, O, and S; wherein each ring optionally isfurther substituted with one or more groups selected from i)-viii) asdefined above;

B) an amine or an amide substituted with at least one substituent asdefined in R3 above;

C) a thiol, a sulfide, a sulfoxide, a sulfone, a thioester or athioamide optionally substituted with one substituent as defined in R3above; or

D) an ether —OR3 wherein R3 is as defined above;

G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR wherein R is (a′) —R″,wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal—OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or(b′) —C(O)R′″ wherein R′″ is as previously defined, and

R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain orbranched C₂-C₉ alkyl which may be substituted at the terminal carbonatom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.

For purposes of this specification C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆alkylthio are intended to include saturated and unsaturated linear,branched and cyclic structures.

Currently more preferred compounds are those wherein G is hydroxy orlower acyloxy and wherein R2 is dimethylheptyl.

According to currently preferred embodiments of the present invention R₁is a heterocyclic moiety selected from the group consisting of animidazolyl, an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, apyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl.

According to further currently preferred embodiments of the presentinvention R₁ is a heterocyclic moiety selected from the group consistingof an imidazolyl, an imidazolinyl, a morpholino, a piperidyl, apiperazinyl, a pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and atetrazolyl, optionally further substituted wherein the substituent isselected from the group consisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆alkylthio, keto, carboxy, nitro, saturated or unsaturated cyclicmoieties or aromatic or heterocyclic moieties wherein each ringcomprises 3-8 carbons interrupted by 1-4 heteroatoms, said heteroatomsin each independently selected from the group consisting of N, O, and S,wherein each ring optionally is further substituted with one or moregroups selected from the group consisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy,C₁₋₆ alkylthio, keto, carboxy, or nitro.

According to more preferred embodiments of the present invention R₁ isselected from the group consisting of imidazole, pyrazole, oxazole,isoxazole, tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine,imidazoline, 2-thio-imidazole, 2-methylthio-imidazoline,4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline, methyl sulfide,methylsulfoxide, acetamido, benzamide, cyano, 1,2,4-triazole,1,3,4-triazole, 1,2,3,4-tetrazole, 1,2,3,5-tetrazole, thiophene, phenyl,morpholine, thiomorpholine, thiazolidine, glycerol, piperazine, andtetrahydropyran.

According to additional more preferred embodiments of the presentinvention R₁ is selected from the group consisting of mono ordi-substituted amines wherein the substituent is selected from the groupconsisting of an C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, imidazolyl,an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, a pyrazolyl,a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, optionallyfurther substituted wherein the substituent is selected from the groupconsisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto , carboxy,or nitro, wherein C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthio areintended to include saturated and unsaturated linear, branched andcyclic structures.

It has been discovered that certain novel compounds of formula (I) aredexanabinol derivatives wherein R₁ is a heterocyclic moiety. Thesecompounds are preferred active agents of the presently claimedcompositions for exhibiting efficient anti-inflammatory properties,including inhibition of prostaglandin synthesis, as well as inhibitionof tumor necrosis factor production, and inhibition of nitric oxideproduction, in addition to providing NMDA receptor blocking andanti-oxidative activity.

It has also been discovered, unexpectedly, that certain novel compoundsof formula (I) are dexanabinol derivatives wherein R₁ is a substitutedamine as defined above. These compounds are preferred active agents ofthe presently claimed compositions for exhibiting efficientanti-inflammatory properties, including inhibition of prostaglandinsynthesis, as well as inhibition of tumor necrosis factor production,and inhibition of nitric oxide production, while being inactive orrelatively inactive as NMDA receptor blockers.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the synthetic scheme used to synthesize certain preferredcompounds designated PRS-211, using dexanabinol as the startingmaterial.

FIG. 2 shows binding curves of compounds according to the presentinvention to the NMDA receptor, as measured by the displacement oflabeled MK-801.

FIG. 3 shows TNF alpha inhibition by novel dexanabinol derivatives.

FIG. 4 illustrates inhibition of PGE2 by novel dexanabinol derivatives.

FIG. 5 shows nitric oxide synthase inhibition by novel dexanabinolanalogs.

FIGS. 6-8 Show the decreased mortality and improved clinical andneurological outcome following transient middle cerebral arteryocclusion in rats treated with certain preferred dexanabinolderivatives.

FIGS. 9-10 Show the ED 50 of certain preferred dexanabinol derivativesin inhibiting inflammation as assessed in the standard ear edema test.

FIG. 11 shows the change in cerebral infarct size in animals treatedwith certain preferred dexanabinol derivatives as assessed in thetransient MCAo test.

FIG. 12 illustrates the improvement in contralateral performance inanimals treated with certain preferred dexanabinol derivatives asassessed in the Staircase test.

FIG. 13 depicts the change in necrotic area in animals treated withcertain preferred dexanabinol derivatives as assessed in the myocardialischemia model.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides novel compounds belonging to the class ofnon-psychotropic cannabinoids, as well as pharmaceutical compositionscomprising these compounds, and methods of using such compounds.Compounds of this class are effective agents for the treatment andprevention of emesis, glaucoma and pain and have been shown to possessneuroprotective and anti-inflammatory properties. It is explicit thatthe present invention excludes known compounds disclosed in U.S. Pat.Nos. 4,876,276, 5,538,993, 5,635,530 and 6,096,740.

The compositions of the present invention are effective to reduce oreven prevent neurological damage, including but not limited toexcitatory amino acid neurotoxicity, due to acute injury or poisoning ofthe CNS, such as injuries due to prolonged seizures, compromised orreduced blood supply, deprivation of glucose supply and mechanicaltrauma, and poisonings by, for example, strychnine, picrotoxin ororganophosphorous compounds.

The compositions of the present invention may also be effective intreating certain chronic degenerative diseases that are characterized bygradual selective neuronal loss. In this connection, the compositions ofthe present invention are contemplated as therapeutically effective inthe treatment of Huntington's chorea, amyotrophic lateral sclerosis,Parkinson's disease and Alzheimer's disease, via mechanisms ofneuroprotection and/or nerve regeneration.

As stated above, the present compositions are of special value inseizures, global hypoxic ischemic insults, in hypoxia, alone or incombination with blood flow reduction (ischemia such as cardiac,unstable myocardial, pulmonary, renal and hepatic ischemias) as well asin cases of cardiac arrest and in cases of abrupt occlusion of cerebralarteries (stroke).

The compositions of the present invention are neuroprotective and mayexert their neuroprotective actions through multiple mechanismsincluding, but not limited to anti-inflammatory and/or antioxidativemechanisms, and some of them are particularly effective in alleviatingand even preventing glutamate neurotoxicity due to acute injuries to thecentral nervous system (CNS), such as injuries due to prolongedseizures, compromised or reduced blood supply, deprivation of glucosesupply and mechanical trauma. The present compositions are alsoeffective in alleviating other damages to the CNS like poison-inducedconvulsions, considered to be associated with amino acid receptors otherthan that of glutamate, for example glycine.

The compositions of the present invention are also effective in thetreatment or prevention of pain, including chronic pain and neuropathicpain.

By virtue of their anti-inflammatory properties it will be recognizedthat the compositions according to the present invention will be usefulin a wide variety of additional indications having an inflammatory orautoimmune mechanism involved in their etiology or pathogenesisexemplified by multiple sclerosis, arthritis such as rheumatoidarthritis and other types of local/general inflammation, encephalitisand HIV-induced neurodegeneration.

Another feature of the present invention is its ability to prevent ortreat the occurrence or growth of gastrointestinal tumors such ascolorectal cancer and colonic polyposis.

A set of the pharmaceutical compositions of the present inventionexhibit inhibitory activity on NOS and cytokines as well as the AA/PGsignaling pathways that regulate or are regulated by COX-2. Thetherapeutic agents of the present invention comprise novel derivativesof non-psychotropic cannabinoids.

The present invention relates to pharmaceutical compositions for thepurposes set out above, in which the active ingredient is a compound ofthe general formula (I):

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer, wherein A—B indicates an optional 1(2) or 6(1)double bond,

R₁ is

A) R3 where R3 is selected from the group consisting of

a) a linear or branched, saturated or unsaturated, carbon side chaincomprising 1-8 carbon atoms interrupted by 1-3 heteroatoms; or

b) a saturated or unsaturated cyclic moiety or an aromatic orheterocyclic moiety having from 5-20 atoms comprising one or two-ringedstructures, wherein each ring comprises 3-8 carbons interrupted by 1-4heteroatoms, said heteroatoms in each independently selected from thegroup consisting of N, O, and S; wherein each ring optionally is furthersubstituted with one or more groups selected from

i) C₁₋₆ alkyl,

ii) C₁₋₆ alkoxy,

iii) C₁₋₆ alkylthio,

iv) Halo,

v) Carboxyl

vi) —CO₂—C₁₋₄ alkyl

vii) keto,

viii) nitro,

ix) a saturated or unsaturated cyclic moiety, or an aromatic or aheterocyclic moiety wherein each ring comprises 3-8 carbons interruptedby 0-4 heteroatoms, said heteroatoms in each independently selected fromthe group consisting of N, O, and S; wherein each ring optionally isfurther substituted with one or more groups selected from i)-viii) asdefined above;

B) an amine —N(R3)₂ or an amide —N(R3)—COR3 substituted with at leastone substituent as defined in R3 above;

C) a thiol —R3SH, a sulfide —SR3, a sulfoxide —SOR3, a sulfone —SO₂R3, athioester —SCOR3 or a thioamide —NC(S)R3, optionally substituted withone substituent as defined in R3 above; or

D) an ether —OR3 wherein R3 is as defined above;

G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR wherein R is (a′) —R″,wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal—OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or(b′) —C(O)R′″ wherein R′″ is as previously defined, and

R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in which R″″ is a straight chain orbranched C₂-C₉ alkyl which may be substituted at the terminal carbonatom by a phenyl group, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.

For purposes of this specification C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆alkylthio are intended to include saturated and unsaturated linear,branched and cyclic structures.

Currently more preferred compounds are those wherein G is hydroxy orlower acyloxy and wherein R2 is dimethylheptyl.

According to preferred embodiments of the present invention R₁ is aheterocyclic moiety selected from the group consisting of an imidazolyl,an imidazolinyl, a morpholino, a piperidyl, a piperazinyl, a pyrazolyl,a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, optionallyfurther substituted wherein the substituent is selected from the groupconsisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto, carboxyor nitro.

According to currently more preferred embodiments R1 is a selected fromthe group consisting of imidazole, pyrazole, oxazole, isoxazole,tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline,2-thio-imidazole, 2-methylthio-imidazoline, 4-methyl-2-imidazoline,4,4-dimethyl-2-imidazoline, methyl sulfide, methylsulfoxide, acetamido,benzamide, cyano, 1,2,4-triazole, 1,3,4-triazole, 1,2,3,4-tetrazole,1,2,3,5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine,thiazolidine, glycerol, piperazine, and tetrahydropyran, optionallyfurther substituted wherein the substituent is selected from the groupconsisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto, carboxy,or nitro, wherein C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthio areintended to include saturated and unsaturated linear, branched andcyclic structures.

In a currently preferred group of compounds, R₂ designates a1,1-dimethylalkyl radical or a 1,2-dimethylalkyl radical with a total ofat least 7 carbon atoms. Also preferred are precursors of suchcompounds. Particularly preferred compounds are those wherein R₂ is1,1-dimethylheptyl or 1,2-dimethylheptyl. It is these embodiments of R₂that are found in THC and its analogues. However, for theneuroprotective activity that characterizes the present invention, it isbelieved that any lower or mid-range alkyl substituent will be suitableat this position.

Throughout this specification, the compounds of the present inventionmay be referred to by their internal reference numbers rather than bytheir full chemical names. The prefix for this series of compounds isPRS-211, followed by a three-digit code for each specific compound ofthe series.

One currently most preferred compound, with which many of thephysiological experiments have been carried out, is the compound, whichmay be referred to as(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(imidazolomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran.This compound is designated hereinafter as PRS-211,095.

Another currently most preferred compound, with which many of thephysiological experiments have been carried out, is the compound, whichmay be referred to as(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(pyrazolomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran.This compound is designated hereinafter as PRS-211,220.

Another currently most preferred compound, with which many of thephysiological experiments have been carried out, is the compound, whichmay be referred to as(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(1H-imidazol-2-ylsulfanylmethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran. This compound isdesignated hereinafter as PRS-211,092.

Another currently most preferred compound, with desirable PGE2 and NOSinhibitory activity, which may be referred to as(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(4-piperidinopiperidinemethyl) 6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran. This compound isdesignated hereinafter as PRS-211,257.

Another currently most preferred compound, with desirable PGE2 and NOSinhibitory activity, which may be referred to as(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(4-methylpiperidinemethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran. This compound isdesignated hereinafter as PRS-211,251.

It is emphasized that all the compounds are of the (+)-(3S,4S)configuration, essentially free of the (−)-(3R,4R) enantiomer, thelatter known to possess the undesired psychotropic side effects. Thus,for example, the enantiomers of the synthetic cannabinoid7-hydroxy-Δ⁶-tetrahydrocannabinol 1,1-dimethylheptyl homolog, have beendescribed [Mechoulam, R., et al., Tetrahedron: Asymmetry 1: 315-319,1990; Mechoulam, R. et al., Experientia 44: 762-764, 1988]. The(−)-(3R,4R) enantiomer, herein designated HU-210, is a highly potentcannabimimetic compound (nearly 100 times more active thanΔ-1-tetrahydrocannabinol, the active component of hashish). The(+)-(3S,4S) enantiomer, herein designated HU-211, also known by thetrivial chemical name dexanabinol, while known to be active as ananalgesic and as an anti-emetic, is inactive as a cannabimimetic even atdoses several thousand times higher than the ED₅₀ of HU-210 (Mechoulam,R. et al., Experientia 44: 762-764, 1988).

As mentioned above, then, the compounds of the general formula (I) asdefined herein are substantially devoid of cannabimimetic centralnervous system activity.

All of the compounds of the present invention are stereospecific (+)enantiomers of the naturally occurring (−) cannabinoids. The (+)stereospecificity provides compounds that are devoid of psychotropicactivity, and have been shown to have substantially no binding to thecannabinoid receptor CB1 of the central nervous system. The IC50 ofbinding of these novel compounds to the CB1 receptor is greater than 300nM, more preferably greater than 1 μM, most preferably greater than 5μM.

Pharmacology

The novel compositions contain in addition to the active ingredientconventional pharmaceutically acceptable carriers, diluents and thelike. Solid compositions for oral administration such as tablets, pills,capsules or the like may be prepared by mixing the active ingredientwith conventional, pharmaceutically acceptable ingredients such as cornstarch, lactose, sucrose, sorbitol, talc, stearic acid, magnesiumstearate, dicalcium phosphate and gums with pharmaceutically acceptablediluents. The tablets or pills can be coated or otherwise compoundedwith pharmaceutically acceptable materials known in the art to provide adosage form affording prolonged action or sustained release. Other solidcompositions can be prepared as suppositories, for rectaladministration. Liquid forms may be prepared for oral administration orfor injection, the term including subcutaneous, transdermal,intravenous, intrathecal, and other parenteral routes of administration.The liquid compositions include aqueous solutions, with or withoutorganic cosolvents, aqueous or oil suspensions, flavored emulsions withedible oils, as well as elixirs and similar pharmaceutical vehicles. Inaddition, the compositions of the present invention may be formed asaerosols, for intranasal and like administration.

The active dose for humans is generally in the range of from 0.05 mg toabout 50 mg per kg body weight, in a regimen of 1-4 times a day.However, administration every two days may also be possible, as the drughas a rather prolonged action. The preferred range of dosage is from 0.1mg to about 20 mg per kg body weight. However, it is evident to the manskilled in the art that dosages would be determined by the attendingphysician, according to the disease to be treated, method ofadministration, patient's age, weight, contraindications and the like.

All the compounds defined above are effective as either NMDA-receptorblockers or oxidative or inflammatory pathway inhibitors and can be usedas active ingredients of pharmaceutical compositions for treatment ofone, or simultaneously several, symptoms of the disorders defined above.The effective dosages are essentially similar, and the more pronouncedeffect is that of NMDA-receptor blocking, in addition to the knowncharacteristics of these compounds. However, it is important to notethat the compounds and compositions of the present invention exhibitgood blocking activity also against convulsants that may not necessarilybe NMDA-receptor mediators. For example, the compositions of the presentinvention can prevent, or at least alleviate, poisoning caused bystrychnine organophosphorous compounds and nitrous oxide.

The compounds of the present invention are administered for theabove-defined purposes in conventional pharmaceutical forms, with therequired solvents, diluents, excipients, etc. to produce aphysiologically acceptable formulation. They can be administered by anyof the conventional routes of administration. The required dose forhumans ranges from 0.005 mg/kg to about 50 mg/kg per unit dosage form.The most preferred dose range is from about 0.1 mg/kg to about 20 mg/kgbody weight.

It will be appreciated that the most appropriate administration of thepharmaceutical compositions of the present invention will depend on thetype of injury or disease being treated. Thus, the treatment of acutehead trauma, stroke or ischemic brain damage resulting from cardiacarrest will necessitate systemic administration of the drug as rapidlyas possible after induction of the injury. On the other hand, diminutionor prophylaxis of chronic degenerative damage, inflammation orgastrointestinal cancer therapy will necessitate a sustained dosageregimen.

PRS-211 compounds convey significant neuroprotection in different invivo models of head trauma, as well as transient and permanent brainischemia. This suggests neuroprotective potential in a wide spectrum ofCNS diseases, poisonings or injuries, as detailed above, includingconditions involving axonal damage such as that sustained in spinal cordinjury. PRS-211 compounds are also particularly useful in treatingneural edema, associated with trauma, infection, tumors or surgicalprocedures including craniotomies and spinal cord manipulation.

Moreover, the combined neuroprotective and anti-inflammatory propertiesof PRS-211 compounds, as well as the known anti-glaucoma properties ofthis class of compounds, leads to special consideration of retinal eyediseases, especially those which are associated with ischemic damage ora hostile biochemical environment. Some non-limiting examples would bediabetic retinopathy, age-related Macular Degeneration, retinal vascularocclusions that are relatively common and may cause considerableischemic damage. All retinal occlusions, venous and arterial, includingthe optic nerve (ischemic optic neuropathy), as well as retinopathy ofprematurity (oxygen toxicity in premature babies), may be included inthis category, as well as any insult that may lead to secondary neuraldamage following direct retinal cell death, e.g., trauma, includingsurgical trauma such as laser burn injuries, inflammations, infectionsand degenerative processes, chronic ischemic damage, includingglaucomatous optic nerve damage and toxic damage (e.g., chloroquinetoxicity) and chronic malnutrition.

The inventors have discovered that certain novel compounds of formula(I), which are preferred active agents of the presently claimedcompositions, such as PRS-211,092, PRS-211,095, PRS-211,128, PRS-211,132PRS-211,220, PRS-211,251 and exhibit combined mechanisms ofneuroprotection and anti-inflammation, including inhibition ofprostaglandin and leukotriene synthesis, as well as the inhibition ofnitric oxide synthesis (as measured by the inhibition of nitric oxidesynthase (NOS))* and the production of cytokines such as tumor necrosisfactor (TNFα) and interleukin-1β, in addition to the NMDA blocking andanti-oxidative activity.

Certain novel compounds of formula (I) such as the majority of the aminederivatives of PRS-211 such as PRS-211,251, PRS-211,253, PRS-211,255 orPRS-211,257 do not exhibit substantial NMDA receptor binding but exerttheir effect via inhibition of the AA/PG pathway and/or asanti-oxidatives. A tabulation of the more preferred compounds accordingthe present invention, is presented in Table 1, whereas their variegatedpatterns of activities in terms of NMDA binding, anti-inflammatory andanti oxidative activities is shown in Table 2. These tables include theknown compound, HU-211 (dexanabinol) for the sake of comparison.

TABLE 1 Chemical Structures of Novel PRS-211 compounds

211,006-000

211,007-000 HU-211

211,041-000

211,044-000

211,047-000

211,092-000

211,095-000

211,102-000

211,103-000

211,118-000

211,119-000

211,128-000

211,132-000

211,133-000

211,134-000

211,145-000

211,159-000

211,204-000

211,211-000

211,212-000

211,220-000

211,251-000

211,253-000

211,255-000

211,257-000

TABLE 2 ACTIVITIES OF PRS-211 COMPOUNDS. Ear Edema NMDA PGE2 TNFα NOSED50 BINDING % inhibition % inhibition % inhibition (μmol/kg) PRS numberIC50 (μM) (at 10 μM) (at 10 μM) (at 10 μM) CO, AA 211,006-000 8 41 46 19104, NA 211,007-000 10 54 33 20  25, 30 HU-211 211,041-000 6 56 23 72 39, NA 211,044-000 6 24 0 211,047-000 100 28 0 211,092-000 >50 57 22 0 28, 32 211,095-000 2.5 72 11 14  29, 27 211,102-000 2.5 211,103-000 3211,118-000 32 211,119-000 62 14 211,128-000 >20 84 48 27  23, 29211,132-000 4.5 61 21 40  70, NA 211,133-000 >100 211,134-000 >100211,145-000 13 63 15 60 211,159-000 >20 87 33 27 211,204-000 10 83 28 85 34, NA 211,211-000 4.6 83 18 0 211,212-000 7.5 96 14 0 211,220-000 0.3547 11 11  57, 10 211,251-000 >100 91 0 37 211,253-000 >100 92 0 27211,255-000 >100 94 0 7 211,257-000 >100 88 10 39

The invention also relates to methods of treatment of the variouspathological conditions described above, by administering to a patient atherapeutically effective amount of the compositions of the presentinvention. The term administration as used herein encompasses oral,parenteral, intravenous, intramuscular, subcutaneous, transdermal,intrathecal, rectal and intranasal administration.

Binding studies show that certain embodiments of the present inventionblock NMDA receptors in a stereospecific manner, and that theinteraction occurs at binding site(s) distinct from those of some othernon-competitive NMDA antagonists or of glutamate and glycine. This, andthe other compounds according to formula (I), may therefore prove usefulas non-psychoactive drugs that protect against NMDA-receptor-mediatedneurotoxicity.

Test Systems

Evaluation of the therapeutic effects of the novel PRS-211 compounds hasnow been carried out in a series of experimental systems to support theutility of these drugs as neuroprotectants, anti-inflammatory agents andanti-oxidative agents. These effects have been evaluated both in vitroand in vivo, and have been corroborated utilizing the following systems:

(a) Binding to the NMDA receptor linked channel: Non competitiveantagonists of NMDA, the primary example of which is the compoundMK-801, bind to a site within the NMDA receptor channel thus preventingthe activation of the receptor-channel complex and the consequentneurotoxicity. The ability of various compounds to compete with thebinding of tritium labeled MK-801 to brain membranes is considered ameasure of their potency as NMDA non-competitive antagonists.

(b) Inhibition of prostaglandin production: The inhibitory effect of thenew derivatives of Dexanabinol on prostaglandin synthesis is evaluatedin macrophage cell cultures following LPS exposure to induce theinflammatory response. These assays are an indication of theanti-inflammatory activity of the individual analogs.

(c) Inhibition of Tumor Necrosis Factor Alpha: Specific aspects of theinflammatory response cascade are mediated by the cytokine TNFα.Inhibition of TNFα production and/or inhibition of TNFα release by thenew analogs are assayed in macrophage cell cultures activated with LPS.This serves as another indication of the general anti-inflammatorypotential of the novel compounds.

(d) Nitric Oxide Assay: As one aspect of the anti-oxidative potential ofthe novel analogs they were tested for their ability to inhibit theenzyme nitric oxide synthase (NOS). This assay can also serve as anotherindication of the anti-inflammatory cascade, as well as theanti-oxidative mechanisms.

(e) Ear Edema model: The anti-inflammatory activity of the new analogsis screened using an ear edema model in mice. This test system utilizesCroton oil or Arachidonic acid as inflammation inducers. The ability ofthe test compounds to prevent or diminish the inflammatory response tothese stimulants is indicative of their systemic anti-inflammatorycapability.

(f) Improved clinical outcome after closed head injury in rats: Severehead injury is associated with high mortality and severe neurologicalimpairment. Animals subjected to head trauma in a controlled fashionserve as models in which to test drugs of therapeutic potential. Testcompounds can be evaluated both for improved clinical outcome and forreduction of edema induced by closed head injury. The ability ofcompounds to reduce the severity of neurological symptoms and to reducebrain edema is considered a measure of their potency in reducing braindamage.

(g) Transient Middle cerebral artery occlusion (MCAo): The middlecerebral artery is the cerebral blood vessel most susceptible to strokein humans. In animals, coagulation, permanent ligation or permanentplacement of an occluding thread in the artery produces a permanentfocal stroke affecting the MCA territory. Transient ligation orocclusion results in transient focal stroke. Both transient andpermanent focal strokes result in varying degrees of edema andinfarction in the affected brain regions. The ability of compounds toreduce the volumes of edema and infarction is considered a measure oftheir potential as anti-stroke treatment.

(h) Optic Nerve Crush: Application of mechanical pressure to the ratoptical nerve results in crush injury of the axons, which is accompaniedby immediate changes in oxidative metabolism and delayed axonal deathand blindness. The ability of compounds to protect the axons and promoteaxonal sprouting is determined in this assay by following GAP-43 as aspecific protein for nerve growth cones

(i) Parkinson's Disease: An MPTP-induced model of Parkinson's Disease(PD) in mice is used to evaluate the value of the novel PRS-211compounds as therapeutic agents for PD.

(j) Myocardial Protection: A rat model of ischemia and reperfusion wasused to test the ability of compounds to reduce the volumes ofinfarction is considered a measure of their potential ascardioprotectors.

Each of these systems represents an aspect of neuroinflammation,neurotoxicity or ischemia, which is amenable to intervention bypharmaceutical agents. The compounds of the present invention exerttheir demonstrated neuroprotective and anti-inflammatory effects byvirtue of a plurality of mechanisms. Certain embodiments of the presentinvention exert their effect by binding to the NMDA receptor. Among thecompounds of the present invention, the amine derivatives in particularhave been shown to possess little or no NMDA receptor blocking activityand appear to exert their effect via the AA/PG or oxidative pathways.Nevertheless, it cannot be ruled out that their activity is mediated byother receptors or additional mechanisms.

This evaluation clearly supports the concept that PRS-211 compounds arenot acting solely as NMDA receptor antagonists. Rather the therapeuticeffects of PRS-211 compounds may be attributable to additionalmechanisms including inhibition of tumor necrosis factor, antioxidantand radical scavenger properties, anticholinergic action, plateletactivating factor antagonism, anti-inflammatory activity by direct orindirect modulation of arachidonic acid, or inhibition of lipidperoxidation, among others. All of these types of pharmacologic agentshave been suggested potentially to improve functional outcome afterbrain injury. All of these mechanisms may be involved in delayed,secondary or chronic neuronal damage following injury to the CNS(McIntosh, J. Neurotrauma 20:215-243, 1993).

The prototype drug used for evaluation of NMDA blocking activity is thecompound MK-801, which is a potent and selective NMDA receptorantagonist that cannot be used as a human therapeutic agent due to itstoxicity. We have evaluated the similarities and differences between thebiological activities of MK-801 and the novel PRS-211 compounds, assummarized in Table 1 in U.S. Pat. No. 6,096,740, which is incorporatedby reference herein.

Compounds

The currently preferred compounds according to the present invention arenovel analogs of the lead compound dexanabinol, also denoted as HU-211,which is disclosed in U.S. Pat. No. 4,876,276. The neuroprotectiveeffects of dexanabinol are disclosed in U.S. Pat. No. 5,284,867.

Among the novel compounds tested, analogs of dexanabinol bearing aheterocyclic moiety attached via a methylene bridge at position 1 arecurrently more preferred. Some of these novel compounds, particularlythose having shortened tails compared to dexanabinol, as residue R₂,have the added advantage of being more soluble in some aqueoussolutions, whereas the parent compounds are extremely hydrophobic.

These preferred compounds according to the present invention canconveniently be synthesized using dexanabinol as a starting materialaccording to Scheme I, as presented in FIG. 1.

EXAMPLES

The following examples are intended to illustrate the present inventionand these are to be construed in a non-limitative manner.

Synthetic Examples Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(bromomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A suspension of Dexanabinol (1.8 g, 4.66 mmol) and Triphenylphosphine(1.63 g, 6.2 mmol) in acetonitrile (8 ml) was stirred under nitrogenatmosphere at (−10)-(−5)° C. A solution of carbon tetrabromide (2.05 g,6.2 mmol) in acetonitrile (8 ml) was added in portions. After 1 h at˜−10° C. the reaction was allowed to warm to room temperature. Stirringat 18° C. is continued then for 48 hrs. The solvent was evaporated underreduced pressure at 30° C. The residue was diluted with toluene (10 ml)and the obtained solution filtered through a silica gel column (40 gsuspended in toluene), using toluene as eluent. 1.9 g (y=91%) wascollected. Purity was determined by MS, and 1H-NMR.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(imidazolomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A mixture of Compound 1 (100 mg, 0-22 mmole), Imidazole (150 mg, 2.2mmole) and xylenes (2.5 ml) was concentrated under reduced pressure to avolume of about ˜1 ml. The mixture was dissolved in anhydrous THF (4 ml)and the solution stirred at (−)10° C., under nitrogen atmosphere. Butyllithium (0.9 ml, 18 mmole) was added portionwise. The reaction mixturewas allowed to warm slowly to room temperature and stirred for 2.5hours. The reaction was kept overnight at −20° C. The reaction waspoured into crushed ice (˜4 g), neutralized with acetic acid andextracted with ether (3×20 ml). The organic phase was washed with water(20 ml), dried over MgSO₄ and the solvent evaporated. The product waspurified by column chromatography on silica gel (5 g), usingethyl-acetate as eluent. 58 mg (yield=60%) of compound 2 was collectedand purity determined by MS, and 1H-NMR.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(triazolomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A mixture of Compound 1 (100 mg, 0.22 mmole) and 1, 2, 4 triazole wasstirred under nitrogen atmosphere at ˜(−5° C.). DBU (0.26 ml, 1.78 mmol)was added and the reaction mixture left at room temperature overnight.The reaction was poured onto ice, neutralized with acetic acid, andextracted with ethyl-ether (3×10 ml). The organic layer was dried overMgSO₄. On TLC two main compounds were seen and were separated by MPLC:Compound 3a—elution with ethyl-acetate—47 mg Compound 3b—elution withmethanol-ethyl acetate (10:90)—10 mg. Purity was determined by MS, and1H-NMR.

Synthesis of(+)-(S,S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(tetrazolomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A mixture of Compound 1 (0.25 mg, 0.56 mmole) and tetrazole in dry THF(20 ml) was stirred under nitrogen atmosphere at ˜(−) 5° C.(methanol/ice bath). DBU (0.65 ml, 4.4 mmole) was added (in portions),the reaction mixture was allowed to warm slowly to room temperature,left overnight and left for another 24 hours at 40° C. to fully dissolvestarting material. The reaction was poured onto ice, neutralized withacetic acid and extracted with ethyl-ether (3×30 ml). The organic phasewas dried over MgSO₄ and the solvent evaporated. On TLC, two maincompounds were seen, that could be separated by column chromatography onsilica gel (80 gr): Compound 4a (elution with 3% ethyl-acetate intoluene)—110 mg. Compound 4b (elution with 15% ethyl-acetate intoluene)—120 mg. The compounds were washed twice with saturated solutionof NaHCO₃, then with water, dried over Na₂SO₄ and evaporated.

Compound 4a—64 mg. Compound 4b—65 mg

Purity was determined by MS, and 1H-NMR.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(pyrazolomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A mixture of Compound 1 (1 g, 2.2 mmole) and pyrazole (1.50 g, 22 mmol)in anhydrous THF (20 ml) was stirred under nitrogen atmosphere whilebeing cooled. N-Butyl lithium (7 ml, 11 mmol) was added portion wise,and the reaction mixture stirred overnight at room temperature. Thereaction mixture was poured into crushed ice (˜4 g), neutralized withacetic acid and extracted with ethyl acetate (3×20 ml). The organicphase was washed with water (20 ml), dried over MgSO₄ and the solventevaporated. The product was purified by column chromatography on silicagel (5 g), using 10% ethyl-acetate in petroleum ether as eluent toafford 470 mg (yield=60%) of compound Purity was determined by MS, and1H-NMR.

Synthesis of(+)-(3S,4S)-6,6-dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(4,4-Dimethyl-2-imidazolomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

To a solution of Compound 1 (50 mg, 0.4 mmol) in THF,4,4-dimethyl-2-imidazoline (0.4 ml) was added and the mixture stirredovernight at room temperature. The reaction mixture was poured ontocrushed ice, acidified with acetic acid (pH 5), and extracted withEtOAc. The combined organic phase was dissolved in acetonitrile andwater and lyophilized. Yield: 150 mg

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(pyrrolinomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

To a solution of Compound 1 (200 mg, 0.45 mmol) in anhydrous THF (5 ml),pyrroline (0.3 ml) was injected and the mixture stirred at roomtemperature overnight. The reaction was carried out under nitrogenatmosphere. The mixture was poured into ice, acidified with acetic acid(pH 5) and extracted with EtOAc (3×20 ml). The combined organic phaseswere washed with water, dried and evaporated. After lyophilization, 120mg of product was collected.

Synthesis of(+)-(S,S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(pyrrolidinomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A mixture of Compound 1 (0.5 g, 1,1 mmol) and pyrrolidine (0.5 ml) indry THF was stirred at room temperature overnight. The reaction mixturewas poured into water, acidified with acetic acid (pH 6) and extractedwith EtOAc (3×30 ml). The combined organic phase was washed with water,dried and evaporated. The residue was chromatographed over a silica gelcolumn and eluted with 15% methanol in EtOAc. The product obtained waslyophilized to afford 270 mg of pure product.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(methylsulfidomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

To a solution of Compound 1 (100 mg, 0.2 mmol) in THF, sodium methylsulfide was added. The reaction mixture was stirred overnight,evaporated and chromatographed over silica gel to afford 100 mg ofcompound 9.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(methylsulfoxidomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

To a cooled solution (−20° C.) of Compound 1 (1.7 g, 4 mmol) in drydichloromethane (120 ml) meta chloroperbenzoic acid (0.83 g, 4.8 mmol)was added and the mixture stirred 30 min at the above temperature. Thereaction was then poured into a 7% sodium hydrogenocarbonate solution(250 ml) containing excess sulfite. The product was extracted indichloromethane (3×100 ml), washed with water (2×150 ml) dried andevaporated. The residue was chromatographed on silica gel column using5% EtOH in EtOAc. The product obtained was lyophilized to obtain 1.2 gof white powder.

Synthesis of(+)-(S,S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(2-methylthio-2-imidazolinomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

To a mixture of 2-methylthio-2-imidazoline hydriodide (0.3 g, 1.2 mmol)in anhydrous THF (5 ml) DBU (0.18 ml, 1.22 mmol) was added and themixture stirred at room temperature under nitrogen atmosphere. Asolution of Compound 1 (0.2 g, 0.4 mmol) in anhydrous THF (2 ml) wasinjected and the mixture stirred overnight at room temperature. Thereaction mixture was poured into water, acidified with acetic acid (pH5) and extracted with EtOAc. The organic phase was washed with water,dried, with anhydrous sodium sulfate, and evaporated. The residue waschromatographed over silica gel using 25% methanol in ethyl acetate asthe eluting system. 120 mg of compound are obtained.

Synthesis of(+)-(S,S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(phtalimidomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A mixture of compound 1 (1.2 g, 2.15 mmol), phtalimide potassium salt(1.85 g) in methyl sulfoxide (6.0 ml) was stirred under argon at 50 Cfor 18 hours. The resulting mixture was poured onto crushed ice (20 g),acidified to pH 6 with acetic acid, and extracted with chloroform (2×20ml). The extract was dried over sodium carbonate and concentrated underreduced pressure. The residue was subjected to column chromatographyusing toluene and toluene/ethyl acetate as the eluent to afford 0.4 g ofproduct.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(aminomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A solution of Compound 12 (0.51 6 g, 1 mmol) and hydrazine monohydrate(0.159 ml, 3.1 mmol) in ethanol (15 ml) was heated to reflux under argonfor 2 hours. Water (0.5 ml) containing concentrated HCl (0.5 ml, 6 mmol)was added and reflux was continued for an additional hour. The solutionwas left overnight at 22 C. The mixture was diluted with toluene (10ml). The precipitate was filtered off and washed with ethanol (5 ml).The filtrate and the washings were combined, diluted with ethyl ether(10 ml) and washed with 5% sodium hydrogenocarbonate. The organic phasewas separated, dried over sodium carbonate and the solvents wereevaporated under reduced pressure. The residue was crystallized fromacetonitrile to afford 0.3 g.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(acetamidomethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

Compound 13 (0.15 g, 0.39 mmol) was dissolved in acetic anhydride atroom temperature. After about 3 minutes a precipitate formed. Themixture was set aside at 22 C for one hour. Methanol (5 ml) was addedand all precipitate dissolved giving a transparent solution. Thesolution was stored at 22 C for 18 hours and the solvent evaporated. Theresidue was dried in a vacuum oven to give 0.16 g of solid, whichcrystallized from acetonitrile to afford 0.1 g of crystals.

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(1H-imidazol-2-ylsulfanylmethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A mixture of the Compound 1 (2.5 gr, 5.6 mmole) and 2-mercaptoimidazole(2.2 gr, 22 mmole) was stirred under nitrogen atmosphere. Triethylaminewas added slowly and the obtained mixture was stirred overnight at roomtemperature. The reaction mixture was poured into water, neutralizedwith acetic acid and extracted several times with ethyl acetate. Thecombined organic phase was washed with water, dried over MgSO₄ andevaporated to give the crude product which was further purified bysilica gel column (elution with 25% ethyl-acetate in petroleum-ether).After lyophilization, compound 15 was obtained as a white powder (yield1.87 gr, 71%):

Synthesis of(+)-(S,S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(4-(1-pyrrolidinyl)-piperidinemethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A solution of Compound 1 (0.3 g, 0.6 mmol) and4-(1-Pyrrolidinyl)-piperidine (0.2 g, 1.2 mmol) in dry THF (50 ml) wasstirred overnight at room temperature. The reaction mixture was dilutedwith water (30 ml), then acidified with acetic acid and extracted withethyl acetate (3×50 ml). The combined organic fractions were washed withwater, dried with sodium sulfate and evaporated to dryness. The desiredproduct was purified by silica gel column chromatography using ethylacetate-petroleum ether mixture to afford pure product (yield: 85%).

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(4-piperidinopiperidineMethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A solution of Compound 1 (0.3 g, 0. 6 mmol) and 4-piperidinepiperidine(0.21 g, 1.3 mmol) in dry THF (50 ml) was stirred overnight at roomtemperature. The reaction mixture was diluted with water (100 ml), thenacidified with acetic acid and extracted with ethyl acetate (3×50 ml).The organic phase was washed with water (50 ml), dried with sodiumsulfate and evaporated to dryness. The desired product was purified bysilica gel column chromatography using ethyl acetate-petroleum ethermixture to afford pure product (yield 78%).

Synthesis of(+)-(S,S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(N-benzylanilineMethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

A solution of Compound 1 (0.3 g, 0.6 mmol) and N-Benzylaniline (0.23 g,1.3 mmol) in dry THF (50 ml) was stirred overnight at room temperature.The reaction mixture was diluted with water (100 ml), then acidifiedwith acetic acid and extracted with ethyl acetate (3×50 ml). The organicphase was washed with water (50 ml), dried with sodium sulfate andevaporated to dryness. The desired product was purified by silica gelcolumn chromatography using ethyl acetate-petroleum ether mixture toafford of pure product (yield: 86%).

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(N-(2-Aminoethyl)pyrrolidinemethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

Compound 19 (0.5 g, 1.3 mmol) and N-(2-Aminoethyl)pyrrolidine (0.44 g,3.9 mmol) were dissolved in methanol (30 ml) and stirred at roomtemperature for one hour. Sodium cyanoborohydride (0.72 g) was added andthe reaction stirred at room temperature overnight. The reaction mixturewas acidified with diluted HCl (1N) and extracted with Ethyl acetate(3×50 ml). The organic fraction was washed with water (50 ml), driedover anhydrous sodium sulfate and evaporated under vacuum. The residuewas chromatographed over silica gel column using ethyl acetate—petroleumether as the eluting system to afford the desired product (yield: 69%).

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(N-diethanolaminemethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

Compound 19 (0.5 g, 1.3 mmol) and diethanolamine (0.35 g, 3.3 mmol) weredissolved in methanol (30 ml) and stirred at room temperature for onehour. Sodium cyanoborohydride (0.72 g) was added and the reactionstirred at room temperature overnight. The reaction mixture wasacidified with diluted HCl (1N) and extracted with Ethyl acetate (3×50ml). The organic fraction was washed with water (50 ml), dried overanhydrous sodium sulfate and evaporated under vacuum. The residue waschromatographed over silica gel column using ethyl acetate—petroleumether as the eluting system to afford the desired product (yield: 69%).

Synthesis of(+)-(3S,4S)-6,6-Dimethyl-(1,1-dimethylheptyl)-1-hydroxy-9-(4-methylpiperidineMethyl)-6a,7,10,10a-tetrahydro-6H-dibenzo[b,d]pyran

Procedure

4-methyl piperidine (0.47 ml, 6 mmole) was added to a solution ofCompound 1 (1.0 gr, 2.24 mmol) in dry THF (50 ml) and the obtainedmixture was stirred overnight at room temperature. The reaction mixturewas poured into water, neutralized with acetic acid and extractedseveral times with ethyl acetate. The combined organic phases werewashed with water, dried over anhydrous MgSO₄ and evaporated to give acrude product which was purified by flash silica gel columnchromatography (elution with 25% ethyl-acetate in petroleum-ether).After lyophilization, compound 22 was obtained as a white powder (0.62g).

PHYSIOLOGICAL EXAMPLES Physiological Example 1 PRS-211 Analogs Analyzedby Radioligand Binding Studies

The identification of possible recognition sites for PRS-211 analogs wascarried out by measuring the ability of these PRS-211 analogs to inhibitthe binding of MK-801 to rat forebrain membranes. Radioligand bindingstudies demonstrated that the parent compound HU-211 competes with thebinding of MK-801 to membranes, while it is unable to inhibit AMPA orkainic acid binding.

Forebrain membrane preparation: Brains were removed from Sprague-Dawleyrats no more than 5 min after decapitation. Membrane preparations wereisolated according to a procedure described previously (Eshhar et al.,Brain Res. 476:57, 1989). Prior to radioligand binding measurements,endogenous glutamate present in membranes was removed from thepreparation by subjecting the membranes to 3-4 successive washings in 10mM Tris HCl pH 7.2, performed at 4° C.

Radioligand binding studies: The specific binding of the new analogs toNMDA receptors was determined by their ability to displace (³H)MK-801from NMDA receptor in rat forebrain preparations. Specifically,rat-forebrain membranes (0.1 mg) were incubated for 2 hours at roomtemperature with 10 nM of tritiated MK-801 and with the PRS-211compounds at 3 doses each. Non-specific binding was determined by theuse of 0.1 mM MK-801. Following the incubation, bound radioligand wasseparated from unbound by filtration through GF/B filters. The filterswere counted in a β-counter and log of analog concentrations versus % of(³H)MK-801 specific binding was plotted. The IC₅₀ was calculated fromthis plot.

Binding of [³H]MK-801 to membranes was carried out in the presence of 30μM glycine and 10 μM L-glutamate. Membranes (250 μg protein) wereresuspended in 50 mM tris-acetate pH 7.4 buffer and incubated with 10 nM[³H]MK-801, either alone or in the presence of PRS-211 compounds at0.1-100 μM concentrations for three hours at room temperature (RT).Reaction buffers used in the different radioligand binding studiescontained 10% of an ethanol/Emulphor 620/deionized water mixture. Theratio (by volume) of the respective components in the mixture was20/3/57. This mixture is required for solubilizing PRS-211 compounds atconcentrations above 30 μM. Reaction volume was 1 ml. Non-specific[³H]MK-801 binding was determined in the presence of 100 μM unlabeledMK-801.

Concentration dependence of certain PRS-211 analogs on inhibition of[³H]MK-801 binding is illustrated in FIG. 1. The inhibition constant(K_(I)) value displayed by PRS-211,007 was found to be 11.0±1.3 μM. TheIC₅₀ of several dexanabinol derivatives is presented in FIG. 2. Thus,for example, the mean IC50 of HU-211 (PRS-211,007) was 10 μM comparedwith that of 2.5 μM for PRS-211,095 (imidazole derivative), versus >20μM of PRS-211,128 and 4.5 μM for PRS-211,132 and 0.35 μM of PRS-211,220(pyrazole derivative).

Physiological Example 2 The in Vivo Anti-inflammatory Effect of PRS-211Compounds in the Ear Edema Model

The anti-inflammatory activity of the new analogs was established in theear edema model in mice using Croton oil (CO) or Arachidonic acid (AA)as inflammation inducers.

Briefly, the animals were anesthetized and either test analog oridentical volume of vehicle is injected IP. CO or AA solution dilutedwith acetone was injected (ear/ear) to one ear. The contralateral ear,serving as control, received an equal volume of the diluent. One to 3hours post injection the animals were euthanized and ear thicknessmeasurements are taken in duplicates using a low tension, spring loadeddial micrometer. The edge of the micrometer pads was placed on the outeredge of the ear. Thickness was measured in units of 0.01 mm. Tissueweight was determined by excising a 6 mm diameter disc of ear tissuefrom the ear lobes using a metal punch. Inflammation/Edema is expressedas the increase in thickness/weight of treated versus diluent treatedcontralateral ear in animals injected with either test new analogs orvehicle. Dose response curves, with at least 4 doses, are used tocalculate potency (ED₅₀), and the maximal efficacy (% of inhibition ofEdema), for each of the test drugs.

Physiological Example 3 In Vitro Screening of Anti-inflammatory Activity

A. Inhibition of Prostaglandin Synthesis

The inhibitory effect of Dexanabinol new analogs on prostaglandinsynthesis was evaluated in macrophage cell cultures. Macrophages wereseeded in a 24 well NUNC plates, and incubated with DMEM medium for 24hours to allow attachment to plastic. The wells were vacuumed and adifferent new analog is added for 1 hour. Treatments are done intriplicates. Following, LPS was added for a duration of 3-24 extrahours, to induce the inflammatory response. The supernatants werecollected and analyzed for PGE₂ by enzyme immunoassay technique Biotrakkit (Amersham Pharmacia Biotech). The results obtained with certainpreferred novel analogs are summarized in TABLE 1 above.

B. Inhibition of TNFα

The method for macrophage cell culture growth is identical to the onedescribed in PGE2 assay. Aliquots of the supernatants collectedfollowing stimulation with LPS were quantitated for TNFα by enzymelinked immunosorbent assay (ELISA) using HRP conjugated to anti-TNFαantibodies and peroxidase as a substrate for the colorimetric reaction.The peroxidase catalyzed color reaction was stopped by acidification andthe absorbance at 450 nm is measured. The absorbance at this wavelengthis proportional to the concentration of TNFα in the sample determinedfrom a standard curve plotting the concentrations of TNFα standardsversus their absorbance.

C. Inhibition of Nitric Oxide Synthase (NOS)

The final products of NOS are Nitrite (NO₂) and Nitrate (NO₃). The invitro fluorimetric assay provides an accurate method for the measurementof the Nitrite, which is the majority of NO products. The principle ofthe fluorimetric assay is based on the addition of DAN(2,3-diaminonaphthalene) to the aliquots of the supernatants collectedfrom macrophage cell cultures incubated with the new analogs andstimulated with LPS. Following, NaOH was added to convert Nitrite to afluorescent compound 1(H)-naphthtriazole. The fluorescence was measuredimmediately in a fluorimeter using excitation wavelength of 365 nm andan emission wavelength of 450 nm.

Physiological Example 4 The Effect of PRS-211 Compounds on CerebralEdema in a Rat Model of Closed Head Injury

The cerebroprotective effect of PRS-211 compounds was assessed in amodel of head trauma (HT) in rats. Injury was induced in anesthetizedrats by a weight-drop device followed by a recovery period of up to 48hours. This type of trauma produces brain edema (i.e. increase in watercontent, decrease in specific gravity in the brain), breakdown of theblood brain barrier (BBB) and clinical dysfunction. The clinical statusof the rats was evaluated 1, 24 and 48 hours after injury along withmeasuring the extent of cerebral edema. The neurological deficit,assessed by a set of criteria termed the Neurological Severity Score(NSS), is maximal at 1 hour after the initiation of head trauma. The NSSslowly decreases over time from the initiation of HT, with the gradualspontaneous recovery of the rats.

The novel analog PRS-211-095 significantly reduces edema formation andBBB disruption when given before (30 min), immediately after HT (0 min)or even 1 and 2 hours after HT.

The doses required for significant neuroprotection depend on the mode ofadministration and range from 0.5-20 mg/kg. It is also important to notethat the NSS, mainly specific motor function (e.g. beam-walk andbalance) improved significantly upon administration of PRS-211. In fact,even one dose of 5 mg/kg of PRS-211-095, given 1 hour after the impact,effectively reduced edema and improved the clinical outcome measured 24hours after HT.

Experimental procedure: The model was described in detail by Shapira etal., Crit. Care Med. 16:258-265, 1988. Rats were subjected to headtrauma (HT) by a weight-drop device and surviving rats were followed upafter one week. During that period they had free access to food andwater, and were kept 2-3 rats to a cage. At any predesignated time (15min, 1, 4, 24, 48 hrs, etc.) rats were sacrificed. Their brains werethen rapidly removed and cortical tissue taken to determine watercontent, ions and the metabolites of interest at any particularmetabolic cascade studied. During the recovery period, the clinicalstatus was evaluated by a set of criteria (NSS).

Trauma induced a significant decrease in specific gravity (SG) of braintissue and increase in water content following head injury. Edemadeveloped since more water accumulates in either the extracellular(vasogenic) or intracellular (cytotoxic) spaces. The methods employed todetermine edema are based on linear gradient columns of bromobenzene andkerosene (for SG) and for water content on drying the tissue in adesiccated oven. Tissue pieces (20 mg each) were placed on top of thecolumn and the SG calculated from the equilibrium position in thecolumn, using a standard curve.

Results

Table 3 summarizes the results of a typical experiment in which thenovel analog PRS-211-095 was injected at doses of 5-10 mg/kg. The drugwas given half an hour before, or one hour after, the induction oftrauma and its effect on edema and clinical outcome was evaluated 1 and24 hours later. The results indicates a significant (p=0.003) decreasein the degree of edema developed after head trauma (CHI), as well as ahighly significant decrease (p<0.001) in the neurological deficit scoreas a result of PRS-211-095 treatment to traumatized rats.

TABLE 3 Cerebroprotective effects of HU-211 and PRS-211,095 in ratsfollowing Closed Head Injury (CHI) Neurological N = Water content scoreΔNSS no. of Treatment Left Right 1 hr 24 hr 24 hr rats Untreated 84.82 ±0.33 79.44 ± 0.32 12.2 ± 0.6 8.7 ± 0.6 3.5 ± 0.31 13 CHI-controlDexanabinol 83.09 ± 0.51 79.37 ± 0.30 11.7 ± 0.7 6.3 ± 0.6 5.5 ± 0.42  8HU-211 *p = 0.007 PRS-211,095 82.89 ± 0.48 79.75 ± 0.20 11.9 ± 0.6 5.4 ±0.5 6.5 ± 0.45 10 *p = 0.003

The effect of PRS-211 analogs was calculated by the percent edemaformation, where 100% was taken as edema in control, non-treated rats.Thus, the reduction in the SG was calculated as follows:

SG (sham)−SG (drug)/SG (sham)−SG (cont)×100

The increase in water content was calculated as follows:

[%H₂O(drug−%H₂O(sham)/%H₂O(cont)−H₂O(sham)]×100

All results presented in the table are statistically different (p<0.05)from control, traumatized vehicle treated rats.

After we established the effect on edema, when given 30 minutes priorto, or right after, HT, we investigated the “therapeutic window,” namelyPRS-211, 25 mg/kg i.p. was given one, two or three hours after HT. Itseffect on NSS (and on specific motor function) was assessed, as well asthe effect on edema and BBB integrity. FIGS. 6-8 summarize the resultsof these studies. As can be seen, PRS-211 was fully effective, even whenadministered up to 2 h post-injury; at 3 h post-trauma the effect wasless pronounced.

Conclusion

Severe head injury, or cerebral ischemia, is associated with a highmortality rate (exceeding 50%) and poor functional outcome. Despiteextensive clinical and experimental research, there are no well-definedtherapies for these conditions. There are very few available treatmentsfor brain injury today and the gradual progressive biochemical changesthat occur after head trauma can lead to the evolution of permanentneuronal damage. The results clearly demonstrate that the compounds ofthe instant invention, namely PRS-211 compounds possesscerebroprotective properties in a model of closed head injury.

Physiological Example 5 Neuroprotection by PRS-211 Compounds inTransient Middle Cerebral Artery Occlusion (MCAo), Infarct SizeEvaluation

Experimental design. The design was a randomized one, performed in amasked fashion as to whether drug or vehicle was being given, and anattempt was made to generate approximately equal numbers of drug- andvehicle-treated animals.

Materials

a. Male rats (8/treatment group) 320-380 gr. (Harlan Israel).

b. Halothane (Rhone Poulenc France)

c. Pentobarbitone (Pental Veterinary, CTS Israel).

d. Poly-L-Lysine (Sigma, USA).

e. Silk suture material 3-0 and 4-0.

f. Nylon (Polyamid) suture material 3-0. Four cm pieces were cut andpositioned in a solution of 1% Poly-L-Lysine for 1 minute and dried inan oven (60° C.) for 60 minutes. The tip of each piece was rounded undera flame.

g. Saline (Teva Medical).

h. Blank cremophor ethanol cosolvent (Pharmos).

i. Dexanabinol in cremophor ethanol cosolvent 50 mg/ml. (BothDexanabinol and its vehicle were diluted in saline prior to drugadministration.

j. Analogs PRS-211,092, PRS-211,095, PRS-211,128 PRS-211,132 andPRS-211,220 in cremophor ethanol cosolvent, 50 mg/ml.

k. The analogs were diluted in saline prior to drug administration.

Methods Transient MCAo

a. Surgical details of the MCAo method used for testing dexanabinolanalogs, are presented in Belayev, et al., (Belayev, Busto, Weizhao, andGinsberg, Stroke 26:2313-2319, 1995) which is incorporated herein in itsentirety by reference.

b. Two hours after the start of MCAo, the animals were reanesthetizedwith halothane, the neck wound was re-opened and the nylon thread pulledout of the Internal carotid artery (ICA). The skin wound was then closedwith 3-0 silk suture material and the animals allowed to recover fromthe anesthesia.

c. Three sets of animals were tested. In the first test immediately (2+0hours) before thread removal the animals were injected IV with eitheranalogs PRS 211,095, PRS 211,128, PRS 211,132, or Dexanabinol (PRS211,007) each at a dosage of 5 mg/kg. One group of animals was treatedwith 5 ml/kg vehicle alone. In another group, the animals subjected to“sham” operation, the suture was passed into the ICA, as describedabove, but immediately withdrawn. In the second set dexanabinol,PRS-211,092, PRS-211,095 and PRS-211,220 were used, at 2+0 hours. In thethird set dexanabinol, PRS-211,092, PRS-211,095 and PRS-211,220 wereadministered 1 hour after thread removal (2+1 hours). Laser Doppler Flow(LDF) determined the success of MCAo—a drop of more then 50% in cerebralblood flow was considered as a sign of successful MCAo. Although LDF wasa helpful corroborative sign, clinical outcome remained the finalinclusion/exclusion criterion, since, with the exception of thePRS-211,220 staircase study, LDF was introduced only after initiation ofthe study.

Behavioral/neurological Outcome Assessment

A detailed investigation of neurological performance was carried out onthe first, third and the 7^(th) day after the MCAo. Two parameters wereexamined: posture and the flexion reflex (based on Nokon and Chuang,NeuroReport 9, 1998: 2081-2084). Animals were scored according to theirperformance:

Posture Score

0-Normal

1-Slight twisting

2-Marked twisting

3-Marked twisting and forelimb flexion

Flexion Reflex Score

0-Normal

1-Slight deficit

2-Moderate deficit

3-Severe deficit

Morphological Assessment of the Infarct Size

One week after the ischemic insult, animals were euthanized withpentobarbitone 100 mg/kg IP. The animals were perfused through the heartwith heparinized 4% formaldehyde solution in PBS (pH 7.4). Brains werethen removed, and kept in the same solution before preparation forhistological evaluation of the brain infarct volume.

Statistical Analysis

The infarct size was compared using ANOVA (analysis of variance)followed by Duncan's post hoc test.

Results

a. Mortality rate

No mortality was detected in the sham and 211,095 treated rats. A lowmortality rate (9%) was seen in the Dexanabinol treated animals. Themortality rate among the other treatment groups (vehicle, 211,128 and211,132) was similar (around 25%).

b. Behavioral/Neurological Outcome

Animals treated with analog PRS-211,095 demonstrated fewer neurologicaldeficits and recovered faster compared to the other treatment groups.

c. Neuropathology

The means of the infarct volume and the percentage relative size to thecontralateral hemisphere were least in animals treated with PRS-211,095,PRS-211,092 and PRS-211,220, the latter at 0.5 mg/kg, followed by thevalues in animals treated with 211,132. The reduction of infarct sizewas 60%, 52% and 48% for PRS-211,095, PRS-211,092 and dexanabinol(PRS-211,007), respectively.

Conclusions

These results show the novel analogs PRS-211,095, PRS-211,092 andPRS-211,220 are potent both in terms of preservation of function andreduction of brain lesion size after MCAo. These data can possibly beinterpreted in the context of the relatively high affinity ofPRS-211,095 and PRS-211,220 for the NMDA receptor as well as beingpotent inhibitors of COX-2. In contrast, PRS-211,128 has no detectableaffinity for the NMDA receptor and was inactive in terms of function,reduction of mortality and brain morphometry. These structure-activityrelationships suggest that affinity for the NMDA receptor is animportant, but not the only, component for neuroprotection againstischemia. PRS-211,092 shows low affinity for the NMDA receptor andsuggests that other, unidentified mechanisms are important forneuroprotection.

Physiological Example 6 Neuroprotection by PRS-211 Compounds inTransient MCAo: Evaluated by the Staircase Test

The test challenges fine motor, sensory and stereognostic function ofthe cortex in enabling the forepaw (hand) to identify, grasp andaccurately manipulate small objects such as food pellets. Loss of theability to identify, grasp and manipulate small objects reflects alesion of the fronto-parietal cortex and is a common and cripplingdeficit after stroke in humans.

Materials

The materials for this procedure are identical to those fromPhysiological Example 5, except for the different concentrations of PRS211,095. The procedural differences are as follows:

Male Sprague Dawley rats, 230-270 gr (Harlan, Israel) were used. Thefollowing compounds and their respective concentrations were tested:Dexanabinol and analogs PRS-211,095 and PRS-211,220 in PEG ethanolcosolvent 50 mg/ml. Dexanabinol, the analogs and their vehicle werediluted in Intralipid® 20% (Pharmacia) prior to drug administration.

Methods

1. Training for Functional Evaluation

The procedure is essentially as described in Montoya et al. (Montoya,Campbell-Hope, Pemberton and Dunnet, J. Neurosci. Meth. 36:219-228,1991) Animals were kept under mild food deprivation for 3-5 days,receiving 15-gr. food once a day (between 16:00 and 17:00) and freeaccess to water. Animals were trained prior to the test for 3-5 daysaccording to the protocol of Sharkey et. al. (Sharkey, Crawford, Butcherand Martson, Stroke 27:2282-2286, 1996).

In brief, the staircase box contains two rows of seven stairs in each.Two 45 mg food pellets were placed on each step. Animals were placed inthe box for 15 minutes sessions. At the end of the session the number ofeaten (grasped) and displaced pellets was counted and recorded. Animalswere tested twice a day, 4 hours apart. The first session was performedbetween 8-9 AM and second between 12-13 PM. The animals were traineduntil they grasped (ate) at least 8 pellets from each set of stairs fortwo consecutive sessions. Up to one week after the end of the successfultraining session the animals underwent transient MCAo. Transient MCAowas performed in essentially the same manner as described inPhysiological example 5 with the following modification at step i:

Two minutes prior to thread removal rats were re-anesthetized andadministered IV with Dexanabinol 5mg/kg, analogs

PRS-211,220 or PRS-211,095 at 0.5, 2.5, 5 or 10 mg/kg or vehicle 5ml/kg. One additional group was sham-treated.

2. Functional Assessment Following Transient Focal Ischemia

Animals were allowed to recover from the surgery for five days. The mildfood deprivation was re-employed during the days following the ischemicinsult. They were tested in the staircase box using the above method for9-12 days. Animals were weighed twice a week during this period.

3. Morphological Assessment of the Infarct Size

At the end of the evaluation period, animals were euthanized withpentobarbitone 100 mg/kg IP. The animals were perfused through the heartwith heparinized 4% formaldehyde solution in PBS (pH 7.4). Brains werethen removed, and kept in the same solution for at least 24 h. Then thebrains—from the rostral side of the cortex to the cerebellum—were sunkin 30% sucrose in PBS, cryosectioned (20 μm), dried and stained withthionin for histological evaluation of the brain infarct volume. Eightsections at the levels of: 3.3;+2.8;+1.8;+0.8;−0.4;−1.4;−2.2; and −3.4from Bregma were measured (Swanson, Rat Brain Atlas, 1992). Sectionswere captured by a CCD camera (V-tech MP-470) and image analyzed (Scionimage 1.62A). Infarct size was determined from the series of the 8sections by either calculation of the infarct volume (mm3) by summingthe series of mean lesion areas of two adjacent sections multiplied bythe distance between them or calculation of the mean infarct size aspercentage of the contralateral hemisphere size; calculation of the meanof the surviving ipsilateral side as percentage of the contralateralhemisphere size; calculation of the mean ipsilateral side ventricle aspercentage of the contralateral hemisphere ventricle.

Statistical Analysis

a. The infarct size was compared using ANOVA (analysis of variance)followed by Duncan's post hoc test.

b. The staircase task performance was evaluated after collecting thedata and compressing it into four blocks of six trials: First block—Thelast day of preoperative testing Second block—Tests from day 6, 7 and 8post ischemic insult Third block—Tests from day 9, 10 and 11 (or 12 or13) post ischemic insult (a pool of the second 3 days) Fourthblock—Tests from day 14-18 post ischemic insult

c. Data were analyzed using ANOVA (analysis of variance) followed byTukey's post hoc test.

Results

a. Physiological Parameters

No differences in the two measured physiological (blood glucose andrectal temperature) among the different treatment groups or at differenttreatment times were detected.

b. Mortality Rate

Mortality in the present study was low as can be seen in Tables 4 and 5.

TABLE 4 Mortality rate for Dex and PRS-211,095 treated animals No. DeadTreatment N Animals Mortality Rate (%) Sham 13 0 0 Vehicle 13 1 8 Dex 5 8 0 0 211,095 0.5 14 3 21  211,095 2.5 10 3 30  211,095 5  9 0 0211,095 10 11 1 9

TABLE 5 Mortality rate for Dex and PRS-211,220 treated animals No. DeadTreatment N Animals Mortality Rate (%) Sham 18 0 0 Vehicle 25 4 16211,220 0.1 16 4 25 211,220 0.5 16 3 18.75 211,220 2.5 13 4 30.7 211,2205 14 2 14 Dex 0.5 11 4 36

c. Body Weight Gain

Animals were kept under mild food deprivation during the staircasetesting (except during weekends). Under these conditions, the followingresults were observed: Sham and PRS-211,095 10 mg/kg treated rats gainedthe maximal body weight (15 and 17% respectively compared to base line).This was statically different (p<0.05) from vehicle-treated rats, whichdid not gain weight at all. (They even lost 4% of their body weight)Dexanabinol and PRS-211,095 (both 5 mg/kg) and PRS-211,220 0.1, 0.5, 2.5and 5 mg/kg treated rats demonstrated a moderate body weight gain (7 and4% for PRS-211,095 and 3, 10, 5 and 4% for PRS-211,220, respectively).These differences were not statistically different. The other groups(PRS-211,095 0.5 and 2.5 mg/kg) did not gain weight.

d. Staircase Test Performance

No significant differences in the ipsilateral performance were observedamong the different treatment groups. The mean number of pelletconsumption was between 8-11 per test. There was a slight decrease inpellet consumption detected in all treatment groups during the firstsession following the ischemic insult (the end of the first week postinsult). This decrease was transient and disappeared during the nextsessions.

A marked impairment was evident in the vehicle-treated rats forcontralateral performance. Pellet consumption was reduced to less than 3in the first session following the ischemic insult. This went up to 5pellets in the third session. PRS-211,095 treated rats demonstrated animproved performance in a dose-related manner. PRS-211,095 0.5 and 2.5mg/kg treated animals showed moderately improved pellet consumption,while the 5 and 10 mg/kg dose levels demonstrated a robust improvement(p<0.05 compared to vehicle). Dexanabinol 5 mg/kg had a similar effect.PRS-211,095 5 mg/kg was superior to the 10-mg/kg dosage and to theDexanabinol only in session 1. No improvement was detected in theDexanabinol 0.5 mg/kg treated rats. PRS-211,220 treated ratsdemonstrated an improved performance in a dose-related manner.PRS-211,220 0.1 2.5 and 5.0 mg/kg treated animals showed moderatelyimproved pellet consumption (20-30% improvement over vehicle), while the0.5 mg/kg dose level demonstrated a robust improvement (more then 60%relative to that of vehicle alone: p<0.05). The best performance in Thelast session (session no. 3) was seen with PRS-211,220 0.5 mg/kg.

A similar phenomenon was detected in the displaced pellets parameter.The vehicle-treated rats displaced the highest number of pellets, whilethe sham-operated rats displaced the least. PRS-211,095 and Dexanabinol5 mg/kg and PRS-211,220 0.5 mg/kg induced an improved performancesimilar to that seen in the previous parameter FIG. 12 depictscontralateral performance in the staircase test. A. Results forPRS-211,095 pellets eaten and pellets displaced and B. Results forPRS-211,220 pellets eaten and pellets displaced.

e. Histopathology

7-8 animals in 3 treatment groups underwent histopathological analysis:Dexanabinol 5 mg/kg, PRS-211,095 2.5 and 5 mg/kg. Under the treatmentconditions, Dexanabinol and PRS-211,095 5 mg/kg reduced the infarctvolume by 35% compared to vehicle. This effect was not statisticallydifferent. No protection was seen with any other dosage.

Conclusions

First, Analog PRS-211,095 and Dexanabinol demonstrated similarneuroprotection following 120 minutes of transient MCAo. This wasevident from the contralateral performance in the staircase as well asfrom the reduction in infarct size. PRS-211,220 at 0.5 mg/kg had themost potent effect in session 3 of the staircase. This is at least 10times more potent than PRS-211,095 and dexanabinol. Second, all doselevels of PRS-211,095 demonstrated improved performance in the staircasetest, but the best activity was seen with the 5 mg/kg dose. It is worthnoting that PRS-211,220 at the 0.5 mg/kg dose performs better thanPRS-211,095 or dexanabinol, both at a 5 mg/kg dose. a. Thehistopathological evaluation revealed that both Dexanabinol and analogPRS-211,095 (both at the 5 mg/kg dose) reduced the infarct size by 35%(compared to vehicle). This difference was not statisticallysignificant, perhaps due to the small number of animals in each group.

Physicological Example 7 Neuroprotection by Dexanabinol Analogs in theMPTP Model of Parkinson's Disease (PD)

The MPTP-mouse model of Parkinson's disease is used to test the efficacyof the compounds to prevent the onset of PD symptoms.

Materials

a. 190 eight week old male C57/BL mice (Harlan Israel).

b. MPTP-HCl (Sigma USA).

c. Saline (TevaMedic Israel).

d. Rat antibody against MAC-1 or F4/80 (Serotec).

e. Rabbit anti-GFAP (Sigma).

f. Rabbit anti-e,n or iNOS (Serotec)

g. Sheep anti-Tyrosine Hydroxylase (TH) (Calbiochem).

h. Secondary antibodies (Zymed)

i. Pentobarbitone sodium 200 mg/m (CTS Israel).

j. Heparin (500 IU/ml, Chaoi France).

k. 4% formaldehyde buffer solution (Frutarom Israel).

Methods

The protocol is based on Liberatore et al. (Liberatore, Jackson-Lewis ,Vukosavic, Mandir, Vila, McAuliffe, Dawson, Dawson and Przedborski,Nature Medicine 5:1403-9, 1999).

The treatment groups are as follows:

Vehicle 5 ml/kg IP once just before MPTP administration.

Dexanabinol or analog 10 mg/kg IP once just before MPTP administration.

Dexanabinol or analog 20 mg/kg IP once just before MPTP administration.

Dexanabinol or analog 20 mg/kg IP once just before MPTPadministration+additional dose 24 hours later.

Table 6 depicts the experimental strategy followed to determine the mosteffective treatment in the MPTP model. 19 groups of animals are treatedas follows:

TABLE 6 Days post MPTP TREATMENTS 24 hours MPTP Saline MPTP + MPTP +MPTP + MPTP + Dex Dex Dex Dex (10 mg) (20 mg) (30 mg) (20 mg × 2) 3 daysMPTP Saline MPTP + MPTP + MPTP + MPTP + Dex Dex Dex Dex (10 mg) (20 mg)(30 mg) (20 mg × 2) 7 days MPTP Saline MPTP + MPTP + MPTP + MPTP + DexDex Dex Dex (10 mg) (20 mg) (30 mg) (20 mg × 2) Naive Dex = dexanbinolor analog

Results

Seven days after MPTP administration the mice were euthanized and theirbrains removed cut and stained using an antiserum to tyrosinehydroxylase (TH-IR), the rate limiting enzyme in monoamainergic neurons.Attention was focused on the Substantia nigra, pars compacta (SNpc),which are comprise of dompaminergic neurons that project to thestriatum. It is these neurons that undergo degeneration in Parkinson'sDisease. MPTP acts as a specific neurotoxin for these neurons, and theaim was to determine whether MPTP-induced nerve degeneration could beameliorated by Dexanabinol at 10 (D-10), 20 (D-20) or 30 (D-30) mg/kg,or 2 injections of Dexanabinol at 20 mg/kg (D-20×2) relative to vehiclealone (V). The results show that MPTP induced a massive decrease in thenumber of TH-IR neurons in the SNpc relative to that in animals treatedwith vehicle alone, and that this decrease was significantly reduced byDexanabinol at 20 and 30 mg/kg.

The rescue effect of the various treatments was calculated by comparingthe number of the TH-IR cells in each group relative to that in animalstreated with MPTP alone, which was arbitrarily assigned a value of zero.The data in Table 7 show that the maximal protective effect ofDexanabinol was obtained at 20 mg/kg.

TABLE 7 Treatments TH-IR SNpc MPTP 0 V −0.19 D-10 −7.85 D-20 28.52 D-3022.73 D-20 × 2 8.5

Physicological Example 8 Optic Nerve Crush Model

The novel compounds are tested in an Optic Nerve Crush model todetermine their effects in axonal survival and regeneration.

Materials

a. Adult, male Sprague Dawley rats 350-550 gr. (Harlan Israel).

b. Dexanabinol or analogs 5% in Cremophore Ethanol (Pharmos).

c. Blank Cremophore Ethanol (Pharmos).

d. Pentobarbitone (Pental Veterinary, CTS, Israel) 1 mg/kg, 1:5 withSaline

e. Xylazine (Vitamed) 1 mg/kg, 1:5 with Saline

f. Antibodies:Mab anti-Gap43 (Sigma, G-9264) Anti-GFAP (Sigma)

Methods

The methodology is as described in Duvdevani et al., (Duvdevani, Rosner,Belkin, Sautter, Sabel, and Schwartz, Rest. Neurol. Neurosci. 2:31-38,1990).

Evaluation of Optic Nerves

At the end of the experiment (8 weeks) the animals are deeplyanesthetized with pentobarbitone 60 mg/kg IP and cardially perfused with4% heparinized formaldehyde solution. The eye and the optic nerves fromthe globe to the chiasm are removed and further fixed by immersion in 4%paraformaldehyde overnight. The optic nerves (from the globe to thechiasm) and the brains (the areas of the lateral geniculate body and thesuperior colliculus) are cryoprotected in 30% sucrose, frozen andsectioned (serial sections: Optic nerves-longitudinal, 16 μm;Brains-coronal, 30 μm). Retinas are prepared as whole mounts. Theretinas and sections are immunohistochemically stained with anti-GAP-43and anti-GFAP. GAP-43 is an acidic, axonally transported membraneprotein present in the CNS and PNS whose presence is indicative ofregenerative growth (Skene and Willard, J Neurosci 1:419-26 1981) whileGFAP, Glial fibrillary acidic protein, is a marker for astrocytes.

GAP-43 positive labeling indicates regenerative growth while GFAPpositive labeling is indicative of glial scar formation. Treatmentsdemonstrating positive labeling for GAP-43 are repeated and processedfor electron microscopy analysis. The number of viable axons in eachtreatment, as a measure for neuroprotection and the number ofunmyelinated, thinly myelinated axons and growth cones, as a measure ofregeneration, are compared in a cross section, 1mm distal from the siteof injury. In animals demonstrating regenerative growth, we measure thelength of regenerating axons.

Physicological Example 9

Rat model of Myocardial Ischemia

The following model of myocardial infarction and heart failure was usedto test the ability of compounds to reduce the volumes of infarction isconsidered a measure of their potential as cardioprotectors.

Materials and Methods

The methods used are essentially as described in Leor and Kloner (Leorand Klonner, Am. J Cardiol. 75:1292-3 1995). The experiment wasperformed in a masked fashion. In brief, Sprague-Dawley rats were giveneither vehicle or PRS-211,095 15 minutes before occlusion. They weresubjected to 45 minutes of coronary occlusion and 4 hours ofreperfusion, after which the coronary artery was reoccluded and 0.25 mlof Unisperse™ dye was injected IV to determine area of risk (AR). Therats were euthanized and hearts were analyzed as to infarct size using a1% solution of triphenylteuazolium chloride for 15 minutes at 37° C. Thearea of necrosis (AN) in each heart was expressed as a percentage of thearea at risk. This was multiplied by the weight of each slice to obtainthe mass of tissue at risk and necrosis.

Results and Conclusions

FIG. 13 shows the area of necrosis per area of risk (AN/AR) for R(PRS-211-095) and S (vehicle) and the area of necrosis of the leftventricle (AN/LV). The Y axis measures the effect on infarct volume inmm³. S is vehicle and R is PRS-211-095. There is a clear decrease ininfarct size in the dexanabinol derivative treated animals.

Although the present invention has been described with respect tovarious specific embodiments thereof in order to illustrate it, suchspecifically disclosed embodiments should not be considered limiting.Many other specific embodiments will occur to those skilled in the artbased upon applicants' disclosure herein, and applicants propose to bebound only by the spirit and scope of their invention as defined in theappended claims.

What is claimed is:
 1. A compound of the general Formula (I):

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer, wherein A—B indicates an optional 1(2) or 6(1)double bond, R₁ is A) R3 where R3 is selected from the group consistingof a) a linear or branched, saturated or unsaturated, carbon side chaincomprising 1-8 carbon atoms interrupted by 1-3 heteroatoms; or b) asaturated or unsaturated cyclic moiety or an aromatic or heterocyclicmoiety having from 5-20 atoms comprising one or two-ringed structures,wherein each ring comprises 3-8 carbons interrupted by 1-4 heteroatoms,said heteroatoms in each independently selected from the groupconsisting of N, O, and S; wherein each ring optionally is furthersubstituted with one or more groups selected from x) C₁₋₆ alkyl, xi)C₁₋₆ alkoxy, xii) C₁₋₆ alkylthio, xiii) Halo, xiv) Carboxyl xv)—CO₂—C₁₋₄ alkyl xvi) keto, xvii) nitro, xviii) a saturated orunsaturated cyclic moiety, or an aromatic or a heterocyclic moietywherein each ring comprises 3-8 carbons interrupted by 0-4 heteroatoms,said heteroatoms in each independently selected from the groupconsisting of N, O, and S; wherein each ring optionally is furthersubstituted with one or more groups selected from i)-viii) as definedabove; B) an amine or an amide substituted with at least one substituentas defined in R3 above; C) a thiol, a sulfide, a sulfoxide, a sulfone, athioester or a thioamide optionally substituted with one substituent asdefined in R3 above; or D) an ether —OR3 wherein R3 is as defined above;G is (a) halogen, (b) C₁-C₅ alkyl, or (c) —OR wherein R is (a′) —R″,wherein R″ is hydrogen or C₁-C₅ alkyl optionally containing a terminal—OR′″ or —OC(O)R′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl, or(b′) —C(O)R′″ wherein R′″ is as previously defined, and R₂ is (a) C₁-C₁₂alkyl, (b) —OR″″, in which R″″ is a straight chain or branched C₂-C₉alkyl which may be substituted at the terminal carbon atom by a phenylgroup, or (c) —(CH₂)_(n)OR′″ wherein n is an integer of 1 to 7 and R′″is hydrogen or C₁-C₅ alkyl.
 2. The compound according to claim 1 whereinR₁ is a saturated or unsaturated cyclic moiety, an aromatic moiety or aheterocyclic moiety having from 5-20 atoms comprising one or two-ringedstructures, wherein each ring comprises 3-8 carbons interrupted by 1-4heteroatoms, said heteroatoms in each ring independently selected fromthe group consisting of N, O, and S; optionally further substituted withat least one substituent selected from the group consisting of loweralkyl, halogen, nitro, cyano, —SR′″, —NHR′″, —N(R′″)₂, —OR′″, —COR′″,—C(O)OR′″ or NH—COR′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl. 3.The compound according to claim 1 wherein R₁ is a heterocyclic moietyselected from the group consisting of an imidazolyl, an imidazolinyl, amorpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, apyrrolidinyl, a triazolyl, and a tetrazolyl, optionally furthersubstituted wherein the substituent is selected from the groupconsisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto, carboxy ,or nitro, wherein C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthio areintended to include saturated and unsaturated linear, branched andcyclic structures.
 4. The compound according to claim 1 wherein R₁ isimidazolyl, pyrazolyl, 2-methyl thio-2-imidazolinyl, or4-methylpiperidine.
 5. The compound according to claim 1 wherein A—B isa 6(1) double bond and G is —OH or lower acyloxy.
 6. The compoundaccording to claim 5 wherein R₂ is 1,1-dimethylheptyl or1,2-dimethylheptyl and wherein R1 is selected from the group consistingof imidazole, pyrazole, oxazole, isoxazole, tetrahydropyridine,pyrazoline, oxazoline, pyrrolidine, imidazoline, 2-thio-imidazole,2-methylthio-imidazoline, 4-methyl-2-imidazoline,4,4-dimethyl-2-imidazoline, methyl sulfide, methylsulfoxide, acetamido,benzamide, cyano, 1,2,4-triazole, 1,3,4-triazole, 1,2,3,4-tetrazole,1,2,3,5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine,thiazolidine, glycerol, piperazine, piperidine and tetrahydropyran,optionally further substituted wherein the substituent is selected fromthe group consisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto,carboxy, or nitro, wherein C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthioare intended to include saturated and unsaturated linear, branched andcyclic structures.
 7. The compound according to claim 1 wherein A—B isabsent and G is —OH or lower acyloxy.
 8. The compound according to claim7 wherein R₂ is 1,1-dimethylheptyl or 1,2-dimethylheptyl and wherein R1is selected from the group consisting of imidazole, pyrazole, oxazole,isoxazole, tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine,imidazoline, 2-thio-imidazole, 2-methylthio-imidazoline,4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline, methyl sulfide,methylsulfoxide, acetamido, benzamide, cyano, 1,2,4-triazole,1,3,4-triazole, 1,2,3,4-tetrazole, 1,2,3,5-tetrazole, thiophene, phenyl,morpholine, thiomorpholine, thiazolidine, glycerol, piperazine,piperidine and tetrahydropyran, optionally further substituted whereinthe substituent is selected from the group consisting of C₁₋₆ alkyl,C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto, carboxy, or nitro, wherein C₁₋₆alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthio are intended to include saturatedand unsaturated linear, branched and cyclic structures.
 9. Apharmaceutical composition comprising as an active ingredient is acompound of the general formula (I):

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer, wherein A—B indicates an optional 1(2) or 6(1)double bond, R₁ is A) R3 where R3 is selected from the group consistingof a) a linear or branched, saturated or unsaturated, carbon side chaincomprising 1-8 carbon atoms interrupted by 1-3 heteroatoms; or b) asaturated or unsaturated cyclic moiety or an aromatic or heterocyclicmoiety having from 5-20 atoms comprising one or two-ringed structures,wherein each ring comprises 3-8 carbons interrupted by 1-4 heteroatoms,said heteroatoms in each independently selected from the groupconsisting of N, O, and S; wherein each ring optionally is furthersubstituted with one or more groups selected from i) C₁₋₆ alkyl, ii)C₁₋₆ alkoxy, iii) C₁₋₆ alkylthio, iv) Halo, v) Carboxyl vi) —CO₂—C₁₋₄alkyl vii) keto, viii) nitro, ix) a saturated or unsaturated cyclicmoiety, or an aromatic or a heterocyclic moiety wherein each ringcomprises 3-8 carbons interrupted by 0-4 heteroatoms, said heteroatomsin each independently selected from the group consisting of N, O, and S;wherein each ring optionally is further substituted with one or moregroups selected from i)-viii) as defined above; B) an amine or an amidesubstituted with at least one substituent as defined in R3 above; C) athiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamideoptionally substituted with one substituent as defined in R3 above; orD) an ether —OR3 wherein R3 is as defined above; G is (a) halogen, (b)C₁-C₅ alkyl, or (c) —OR wherein R is (a′) —R″, wherein R″ is hydrogen orC₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moietywherein R′″ is hydrogen or C₁-C₅ alkyl, or (b′) —C(O)R′″ wherein R′″ isas previously defined, and R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in whichR″″ is a straight chain or branched C₂-C₉ alkyl which may be substitutedat the terminal carbon atom by a phenyl group, or (c) —(CH₂)_(n)OR′″wherein n is an integer of 1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl;together with a pharmaceutically acceptable diluent or carrier.
 10. Thecomposition according to claim 9 wherein R₁ is a saturated orunsaturated cyclic moiety, an aromatic moiety or a heterocyclic moietyhaving from 5-20 atoms comprising one or two-ringed structures, whereineach ring comprises 3-8 carbons interrupted by 1-4 heteroatoms, saidheteroatoms in each ring independently selected from the groupconsisting of N, O, and S; optionally further substituted with at leastone substituent selected from the group consisting of lower alkyl,halogen, nitro, cyano, —SR′″, —NHR′″, —N(R′″)₂, —OR′″, —COR′″, —C(O)OR′″or NH—COR′″ moiety wherein R′″ is hydrogen or C₁-C₅ alkyl.
 11. Thecomposition according to claim 9 wherein R₁ is a heterocyclic moietyselected from the group consisting of an imidazolyl, an imidazolinyl, amorpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, apyrrolidinyl, a triazolyl, and a tetrazolyl.
 12. The compositionaccording to claim 9 wherein R₁ is imidazolyl, pyrazolyl, 2-methylthio-2-imidazolinyl, or 4-methylpiperdine.
 13. The composition accordingto claim 9, wherein A—B is a 6(1) double bond, and G is —OH or loweracyloxy.
 14. The composition according to claim 13 wherein R₂ is1,1-dimethylheptyl or 1,2-dimethylheptyl and wherein R₁ is selected fromthe group consisting of imidazole, pyrazole, oxazole, isoxazole,tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline,2-thio-imidazole, 2-methylthio-imidazoline, 4-methyl-2-imidazoline,4,4-dimethyl-2-imidazoline, methyl sulfide, methylsulfoxide, acetamido,benzamide, cyano, 1,2,4-triazole, 1,3,4-triazole, 1,2,3,4-tetrazole,1,2,3,5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine,thiazolidine, glycerol, piperazine, piperidine and tetrahydropyran,optionally further substituted wherein the substituent is selected fromthe group consisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto,carboxy, or nitro, wherein C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthioare intended to include saturated and unsaturated linear, branched andcyclic structures.
 15. The composition according to claim 9 wherein A—Bis absent and G is OH or a lower acyloxy group.
 16. The compositionaccording to claim 15 wherein R₂ is 1,1-dimethylheptyl or1,2-dimethylheptyl and wherein R1 is selected from the group consistingof imidazole, pyrazole, oxazole, isoxazole, tetrahydropyridine,pyrazoline, oxazoline, pyrrolidine, imidazoline, 2-thio-imidazole,2-methylthio-imidazoline, 4-methyl-2-imidazoline,4,4-dimethyl-2-imidazoline, methyl sulfide, methylsulfoxide, acetamido,benzamide, cyano, 1,2,4-triazole, 1,3,4-triazole, 1,2,3,4-tetrazole,1,2,3,5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine,thiazolidine, glycerol, piperazine, piperidine and tetrahydropyran,optionally further substituted wherein the substituent is selected fromthe group consisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto,carboxy, or nitro, wherein C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthioare intended to include saturated and unsaturated linear, branched andcyclic structures.
 17. The composition according to claim 9 wherein thecarrier or diluent is an aqueous cosolvent solution comprising apharmaceutically acceptable cosolvent, a micellar solution prepared withnatural or synthetic ionic or non-ionic surfactants, or a combination ofsuch cosolvent and micellar solutions.
 18. The composition according toclaim 17 wherein the carrier is (a) solution of ethanol, a surfactant,and water or (b) an emulsion comprising a triglycerides, lecithin,glycerol, an emulsifier, an antioxidant, and water.
 19. A method fortreating or preventing injuries to the central nervous system byadministering to a patient a therapeutically effective amount of apharmaceutical composition comprising a compound of the formula (I):

having the (3S,4S) configuration and being essentially free of the(3R,4R) enantiomer, wherein A—B indicates an optional 1(2) or 6(1)double bond, R₁ is A) R3 where R3 is selected from the group consistingof a) a linear or branched, saturated or unsaturated, carbon side chaincomprising 1-8 carbon atoms interrupted by 1-3 heteroatoms; or b) asaturated or unsaturated cyclic moiety or an aromatic or heterocyclicmoiety having from 5-20 atoms comprising one or two-ringed structures,wherein each ring comprises 3-8 carbons interrupted by 1-4 heteroatoms,said heteroatoms in each independently selected from the groupconsisting of N, O, and S; wherein each ring optionally is furthersubstituted with one or more groups selected from i) C_(1 alkyl,) ii)C₁₋₆ alkoxy, iii) C₁₋₆ alkylthio, iv) Halo, v) Carboxyl vi) —CO₂—C₁₋₄alkyl vii) keto, viii) nitro, ix) a saturated or unsaturated cyclicmoiety, or an aromatic or a heterocyclic moiety wherein each ringcomprises 3-8 carbons interrupted by 0-4 heteroatoms, said heteroatomsin each independently selected from the group consisting of N, O, and S;wherein each ring optionally is further substituted with one or moregroups selected from i)-viii) as defined above; B) an amine or an amidesubstituted with at least one substituent as defined in R3 above; C) athiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamideoptionally substituted with one substituent as defined in R3 above; orD) an ether —OR3 wherein R3 is as defined above; G is (a) halogen, (b)C₁-C₅ alkyl, or (c) —OR wherein R is (a′) —R″, wherein R″ is hydrogen orC₁-C₅ alkyl optionally containing a terminal —OR′″ or —OC(O)R′″ moietywherein R′″ is hydrogen or C₁-C₅ alkyl, or (b′) —C(O)R′″ wherein R′″ isas previously defined, and R₂ is (a) C₁-C₁₂ alkyl, (b) —OR″″, in whichR″″ is a straight chain or branched C₂-C₉ alkyl which may be substitutedat the terminal carbon atom by a phenyl group, or (c) —(CH₂)_(n)OR′″wherein n is an integer of 1 to 7 and R′″ is hydrogen or C₁-C₅ alkyl.20. The method according to claim 19 wherein R₁ is a saturated orunsaturated cyclic moiety, an aromatic moiety or a heterocyclic moietyhaving from 5-20 atoms comprising one or two-ringed structures, whereineach ring comprises 3-8 carbons interrupted by 1-4 heteroatoms, saidheteroatoms in each ring independently selected from the groupconsisting of N, O, and S; optionally further substituted with at leastone substituent selected from the group consisting of C₁₋₆ alkyl, C₁₋₆alkoxy, C₁₋₆ alkylthio, Halo, Carboxyl, —CO₂—C₁₋₄ alkyl, keto, nitro,saturated or unsaturated cyclic moieties or aromatic or heterocyclicmoieties wherein each ring comprises 3-8 carbons interrupted by 1-4heteroatoms, said heteroatoms in each independently selected from thegroup consisting of N, O, and S.
 21. The method according to claim 19wherein R₁ is a heterocyclic moiety selected from the group consistingof an imidazolyl, an imidazolinyl, a morpholino, a piperidyl, apiperazinyl, a pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and atetrazolyl.
 22. The method according to claim 19 wherein A—B is a 6(1)double bond, and G is —OH or lower acyloxy.
 23. The method according toclaim 22 wherein R₂ is 1,1-dimethylheptyl or 1,2-dimethylheptyl andwherein R₁ is selected from the group consisting of imidazole, pyrazole,oxazole, isoxazole, tetrahydropyridine, pyrazoline, oxazoline,pyrrolidine, imidazoline, 2-thio-imidazole, 2-methylthio-imidazoline,4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline, methyl sulfide,methylsulfoxide, acetamido, benzamide, cyano, 1,2,4-triazole,1,3,4-triazole, 1,2,3,4-tetrazole, 1,2,3,5-tetrazole, thiophene, phenyl,morpholine, thiomorpholine, thiazolidine, glycerol, piperazine,piperidine and tetrahydropyran, optionally further substituted whereinthe substituent is selected from the group consisting of C₁₋₆ alkyl,C₁-C₆ alkyloxy, C₁₋₆ alkylthio, keto, carboxy, or nitro, wherein C₁₋₆alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthio are intended to include saturatedand unsaturated linear, branched and cyclic structures.
 24. The methodaccording to claim 19 wherein A—B is absent and G is —OH or loweracyloxy.
 25. The method according to claim 24 wherein R₂ is1,1-dimethylheptyl or 1,2-dimethylheptyl and wherein R1 is selected fromthe group consisting of imidazole, pyrazole, oxazole, isoxazole,tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline,2-thio-imidazole, 2-methylthio-imidazoline, 4-methyl-2-imidazoline,4,4-dimethyl-2-imidazoline, methyl sulfide, methylsulfoxide, acetamido,benzamide, cyano, 1,2,4-triazole, 1,3,4-triazole, 1,2,3,4-tetrazole,1,2,3,5-tetrazole, thiophene, phenyl, morpholine, thiomorpholine,thiazolidine, glycerol, piperazine, piperidine and tetrahydropyran,optionally further substituted wherein the substituent is selected fromthe group consisting of C₁₋₆ alkyl, C₁₋₆ alkyloxy, C₁₋₆ alkylthio, keto,carboxy, or nitro, wherein C₁₋₆ alkyl, C₁₋₆ alkoxy and C₁₋₆ alkylthioare intended to include saturated and unsaturated linear, branched andcyclic structures.
 26. The method according to claim 19 wherein saidcompound is administered in a manner to protect against excitatory aminoacid-mediated neurotoxicity.
 27. The method according to claim 19 whichcomprises administering said compound to a patient who exhibits thesymptoms associated with neural necrosis due to edema, neural necrosisdue to cerebral ischemia, neural necrosis due to head trauma, poisoningof the central nervous system, cardiac arrest, stroke, optic neuropathy,or spinal cord injury.
 28. The method according to claim 19 whichcomprises administering said compound to a patient who exhibits thesymptoms associated with epilepsy, Huntington's disease, Parkinson'sdisease, amyotrophic lateral sclerosis or Alzheimer's disease.
 29. Themethod according to claim 19 wherein A—B designates a 1(2) or 6(1)double bond, R₁ designates a pyrazole an imidazolinyl or an imidazole, Gdesignates hydroxy or a lower acyloxy group and R₂ designates (A) astraight or branched C₆-C₁₂ alkyl radical; (B) a group —O—R³, in whichR³ is a straight or branched C₅-C₉ alkyl radical optionally substitutedat the terminal carbon atom by a phenyl group.
 30. A method for blockingor preventing pain, including chronic and neuropathic pain, orinflammation in a patient which comprises administering to said patient,a therapeutically effective amount of a pharmaceutical compositionaccording to claim
 1. 31. The method according to claim 30 wherein thedaily dosage of said compound is between 0.01 and 25 mg/kg.